WO2017107728A1 - 一种脂肪体的制备方法及其应用 - Google Patents

一种脂肪体的制备方法及其应用 Download PDF

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WO2017107728A1
WO2017107728A1 PCT/CN2016/106732 CN2016106732W WO2017107728A1 WO 2017107728 A1 WO2017107728 A1 WO 2017107728A1 CN 2016106732 W CN2016106732 W CN 2016106732W WO 2017107728 A1 WO2017107728 A1 WO 2017107728A1
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Prior art keywords
protein
fat body
liquid phase
fat
lipid
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English (en)
French (fr)
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刘平生
汪洋
周晓明
马学婧
周畅
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Institute of Biophysics of CAS
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Institute of Biophysics of CAS
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Priority to US16/065,659 priority Critical patent/US10987431B2/en
Priority to EP16877539.3A priority patent/EP3395329B1/en
Publication of WO2017107728A1 publication Critical patent/WO2017107728A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1275—Lipoproteins or protein-free species thereof, e.g. chylomicrons; Artificial high-density lipoproteins [HDL], low-density lipoproteins [LDL] or very-low-density lipoproteins [VLDL]; Precursors thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the invention relates to the field of biotechnology, and in particular to a method for preparing a fat body and an application thereof.
  • Lipid droplet is an organelle composed of a neutral lipid core, a monolayer of phospholipid membrane and related proteins.
  • the functions of this organelle include: 1) synthesis, storage, metabolism and transportation of lipids; 2) storage and degradation of proteins; 3) production and modification of lipid signaling molecules and hormones; 4) interaction with other organelles.
  • lipid droplets have many of the above functions, many human diseases, especially metabolic diseases, are closely related to the formation and dynamic changes of lipid droplets. However, the formation mechanism and dynamic changes of lipid droplets are still difficult. There is still a lack of comprehensive understanding of how lipid droplets form, lipid droplets function and regulatory mechanisms. The complexity of the composition of lipid droplets and their interaction with other organelles interfere with our ability to deconstruct these mechanisms.
  • ALDs artificial lipid droplets
  • lipid droplets there is also a structure in the human body in which a single layer of phospholipid membrane is wrapped with a neutral lipid and covered with a protein, called a lipoprotein.
  • a lipoprotein lipid droplets are very similar, the orientation of the two is different, the surface proteins are different: lipid droplets are present in the cells, and lipoproteins are present in the blood; the major proteins on the lipid droplets are lipid droplets inherent/structural proteins ( Lipid droplet resident/structural protein), and the main protein on lipoprotein is apolipoprotein. Different lipoproteins have different apolipoproteins on their surface. The type and proportion of lipoproteins have a very important impact on human health.
  • High-density lipoprotein is a key component of cholesterol transport in cholesterol. It prevents the deposition of cholesterol on the blood vessel wall and is very important for cardiovascular and cerebrovascular diseases such as atherosclerosis. Preventive effect. Artificial lipoproteins or supplemental preparations or other pharmaceutical carriers that can be used for high density lipoproteins.
  • the problem to be solved by the present invention is to provide a pharmaceutical carrier and a system for studying lipid droplets in vitro.
  • the present invention first provides a method for preparing a fat body composed of a neutral lipid and a monolayer phospholipid film.
  • the method for preparing a fat body comprises the steps of: a1) vortexing a phospholipid and a neutral lipid in a buffer to achieve a reaction between the two, and then performing centrifugation to collect an upper liquid phase from the upper layer. The fat body is separated in the liquid phase.
  • the "separating the fat body from the upper liquid phase” may include the following steps: a2) purifying the upper liquid phase twice or more; each purification may be performed by: the upper liquid phase and the buffer Mixed Then, it is layered, and the upper liquid phase is collected; a3) The upper liquid phase obtained in the step a2) is mixed with the buffer, and then layered, and the lower liquid phase is collected, which contains the fat body.
  • the buffer can be buffer B.
  • the solute of the buffer B and its concentration in the buffer may be: 15 mM to 25 mM HEPES, 80 mM to 120 mM KCl, 1.5 to 2.5 mM MgCl 2 ; the solvent may be deionized water; and the pH may be 7.2 to 7.6.
  • the solute of the buffer B and its concentration in the buffer may specifically be: 20 mM HEPES, 100 mM KCl, 2 mM MgCl 2 ; the solvent may specifically be deionized water; the pH may specifically be 7.4.
  • the parameter of the vortex may be: the total duration is 3 to 5 minutes.
  • the parameters of the centrifugation may be: 18000-22000g, 3-7min.
  • the parameter of the vortex may specifically be: the total duration is 4 min.
  • the parameters of the centrifugation may specifically be: 20000 g, 5 min.
  • the liquid phase of the "upper layer liquid phase is purified twice or more" is mixed with the buffer liquid, and no precipitation is determined after the layering.
  • the "stratification” is achieved by centrifugation, and the parameters of the centrifugation may be: 18000-22000g, 3-7min.
  • the “layering” is performed by centrifugation, and the parameters of the centrifugation may specifically be: 20000 g, 5 min.
  • the "stratification” is achieved by centrifugation, and the parameters of the centrifugation may be: 800 to 1200 g, 3 to 7 min.
  • the “layering” is performed by centrifugation, and the parameters of the centrifugation may specifically be: 1000 g, 5 min.
  • the phospholipid is b1), b2) or b3): b1) 1,2-di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine (DOPC); b2) 1,2- Di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine (DOPC) and 1,2-di-(9Z-octadecenyl)-sn-glycero-3-phosphoethanolamine ( DOPE); b3) 1,2-di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine (DOPC) and 1,2-di-octadecanoyl-sn-glycerol- Choline Phosphate (DSPC).
  • DOPC 1,2-di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine
  • DOPC 1,2-di-octa
  • the neutral lipid is c1) or c2): c1) triglyceride; c2) cholesterol oleate (CO) and triglycerides.
  • 1,2-di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine and 1,2-di-octadecanoyl-sn-glycerol-3- The mass ratio of phosphorylcholine may be 1:0.01 to 2; in the c2), the mass ratio of the triglyceride to the cholesterol oleate may be 1 to 5:1.
  • the mass ratio of glycerol-3-phosphoethanolamine may specifically be 2:1, 1:1 or 1:2; in the b3), 1,2-di-(9Z-octadecenyl)-sn-glycerol-
  • the mass ratio of 3-phosphocholine to 1,2-di-octadecanoyl-sn-glycero-3-phosphocholine may specifically be 2:1, 1:1 or 1:2; in the c2)
  • the mass ratio of the triglyceride to the cholesterol oleate may specifically be 5:1, 4:1, 3:1 or 2:1.
  • a lipolysis liquid chloroform: de
  • the triglyceride may be triolein (TO).
  • TO triolein
  • the triolein (TO) may be specifically a product of Sigma Corporation, catalog number 92860.
  • the mass ratio of the phospholipid to the neutral lipid may be any one of (d1) to (d6): (d1) 0.25 to 3:5; (d2) 3:5; (d3) 2:5 ; (d4) 1:5; (d5) 1:10; (d6) 1:20.
  • the fat body prepared by the above-described method for preparing a fat body is also within the scope of protection of the present invention.
  • the invention also provides a method of preparing artificial lipid droplets.
  • the method for preparing an artificial lipid droplet provided by the present invention comprises recruiting one or several intrinsic proteins and/or functional proteins on a fat body prepared by the preparation method of any of the above fat bodies to obtain an artificial lipid droplet.
  • the intrinsic protein may be an MLDS protein, a MDT-28 protein or a Perilipin-2 protein.
  • the functional protein can be ATGL.
  • the MLDS protein may be e1) or e2): e1) an amino acid sequence such as the protein shown in SEQ ID NO: 2 in the Sequence Listing; e2) a protein represented by e1) substituted with 1 to 10 amino acid residues and/or A protein that has the same function as the MLDS protein is deleted and/or added.
  • the MDT-28 protein may be f1) or f2): f1) an amino acid sequence such as the protein shown in SEQ ID NO: 4 in the Sequence Listing; f2) a substitution of 1 to 10 amino acid residues of the protein represented by f1) / or missing and / or added protein that has the same function as the MDT-28 protein.
  • the Perilipin-2 protein may be g1) or g2): g1) an amino acid sequence such as the protein shown in SEQ ID NO: 6 in the Sequence Listing; g2) a substitution of 1 to 10 amino acid residues of the protein represented by g1) / or missing and / or added protein that has the same function as the Perilipin-2 protein.
  • the ATGL may be h1) or h2): h1) an amino acid sequence such as the protein shown in SEQ ID NO: 8 in the Sequence Listing; h2) a substitution and/or deletion of 1 to 10 amino acid residues of the protein represented by h1) And/or add the resulting protein that has the same function as ATGL.
  • the invention also provides a method of preparing an artificial lipoprotein.
  • the method for preparing an artificial lipoprotein provided by the present invention comprises recruiting one or several apolipoproteins on a fat body prepared by the method for preparing a fat body according to any of the above, to obtain an artificial lipoprotein.
  • the apolipoprotein may be Apo A-I.
  • the Apo A-I can be specifically referred to by Peitsch, M.C. et al. A purification method for apolipoprotein A-I and A-II. Anal Biochem 178, 301-305 (1989). The method described in is obtained.
  • any of (j1) to (j6) is also within the scope of the present invention: (j1) a fat body prepared by the method for producing a fat body according to any of the above, in the preparation of an artificial lipid droplet and/or an artificial lipoprotein and And/or use in a pharmaceutical carrier; (j2) use of an artificial lipid droplet prepared by the preparation method of any of the above-mentioned artificial lipid droplets in preparing a pharmaceutical carrier; (j3) preparation by any of the above-mentioned artificial lipid droplets The application of the artificial lipid droplet prepared by the method in preparing a medicament; (j4) the application of the artificial lipoprotein prepared by the preparation method of any of the above-mentioned artificial lipoproteins in preparing a pharmaceutical carrier; (j5) by any of the above The use of artificial lipoprotein prepared by the method for preparing artificial lipoprotein in the preparation of a medicament; (j6) The use of a fat body prepared by the preparation method of any of the above-mentioned fat bodies as a pharmaceutical
  • the present invention also provides a medicine.
  • the drug provided by the present invention has an active ingredient which is a fat body loaded with a compound having a pharmaceutically useful function; and the fat body is a fat body prepared by the method for producing a fat body according to any of the above.
  • the drug can be a fat soluble drug.
  • the fat-soluble drug may be a drug that is miscible with a neutral lipid.
  • the neutral lipid may specifically be a triglyceride.
  • the drug may specifically be a drug or drug candidate for the treatment of type 2 diabetes and/or metabolic disorders.
  • the compound having medicinal function may be a compound represented by formula a;
  • the compound having a medicinal function may specifically be CDN1163 manufactured by TOCRIS Co., Ltd., and its catalog number is 5869.
  • the "fat body loaded with a compound having a pharmaceutically functional function" is the drug-loaded fat body in Example 3.
  • the preparation method of the drug-loaded fat body can be specifically as follows: (1) 10 mg of TAG is added to a microcentrifuge tube, 100 ⁇ l of chloroform is added, and fully dissolved to obtain a TAG solution; (2) 2 mg of CDN 1163 is taken and placed in another trace. In the centrifuge tube, 200 ⁇ l of chloroform was added and fully dissolved to obtain a drug solution having a concentration of 10 ⁇ g/ ⁇ l; (3) after the steps (1) and (2) were completed, the TAG solution and the drug solution were uniformly mixed, and then high-purity nitrogen was used.
  • step (4) 100 ⁇ l of buffer B and 6 mg of the mixture obtained in the step (3) were added to the tube, vortexed for 4 min (vortexed for 10 s, stopped for 10 s) to obtain a milky white lipid mixture A, and the lipid mixture was centrifuged at 20000 g for 5 min; After that, the bottom part of the microcentrifuge tube is the precipitation component A, and the liquid phase system presents two layers (the upper layer is white with a nail, the white belt with the nail portion is the solution A); (6) after the step (5) is completed, The method of extraction discards the solution A and the precipitated component A, and retains White belt armor, Add 100 ⁇ l of Buffer B, vortex to obtain a milky white lipid mixture B, and centrifuge the lipid mixture
  • Step (7) is the repetition of the step (6), and the number of repetitions of the step (6) in the actual application is based on the absence of precipitation of the white belt under the layer.
  • step (7) After completing step (7), take white with C, add 100 ⁇ l of buffer B, vortex and mix, centrifuge at 1000 g for 5 min, and after centrifugation, the liquid phase system will be layered in two layers (the upper layer is white with diced, white band) The part below D is the solution of Ding). The solution solution is collected, which is a drug-loaded fat body.
  • the invention provides a method for preparing a fat body and an application thereof.
  • the method for preparing a fat body prepareds a fat body composed of a neutral lipid core and a monolayer phospholipid membrane by vortexing and mixing neutral lipids and phospholipids by a two-step method.
  • a fat body composed of a neutral lipid core and a monolayer phospholipid membrane by vortexing and mixing neutral lipids and phospholipids by a two-step method.
  • Recruiting intrinsic and/or functional proteins on fat bodies such as the bacterial protein MLDS, the nematode protein MDT-28, the mammalian protein Perilipin-2, and the adipose triglyceride lipase (ATGL).
  • Lipid droplets recruitment of apolipoproteins such as Apo AI (apolipoprotein AI) on fat bodies to obtain artificial lipoproteins.
  • Apo AI apolipoprotein AI
  • Figure 1 shows the preparation of purified fat bodies using vortexing and two-step centrifugation.
  • A is a fat body preparation flow chart: a is a component for preparing a fat body; b is a flow chart for preparing a fat body, a blue arrow represents a vortex, and a red arrow shows a component to be removed.
  • Figure 2 is a feature of a fat body.
  • C is the ratio of DOPC/total lipid of each component in the process of fat body preparation by thin layer chromatography: a is the result of staining the sample with iodine vapor (lane 1 is the lipid Marker, and lane 2 is the initial preparation component).
  • Lane 3 is a mixed component
  • lane 4 is a fat body a
  • D is the form of fat body and lipid droplets: a is the form of fat body, the left picture is the result of naked eye observation, the middle picture is the observation result of optical microscope, the right picture is the observation result of fluorescence microscope; b is the liver tissue-lip of mouse The form of the drop, the left picture is the result of the naked eye observation, the middle picture is the observation result of the optical microscope, the right picture is the observation result of the fluorescence microscope; c is the form of the brown adipose tissue-lipid droplet of the mouse, and the left picture is the result of the naked eye observation, The picture shows the observation results of the optical microscope, the right picture shows the observation results of the fluorescence microscope; d is the morphology of Rhodococcus erythropolis RHA1-lipid, the left picture shows the results of the naked eye, the
  • Figure 3 shows the factors influencing the preparation of fat bodies.
  • A is the effect of different vortex times (1 is fat body b, 2 is fat body c, 3 is fat body d, 4 is fat body a) on fat body: a is the shape of fat body, b is light of fat body Density, c is the size of the fat body; B is the optical density (a) and size (b) of the fat body prepared by DOPC and TAG in different proportions; C is the optical density of the fat body prepared by DOPC and DSPC in different proportions (a And size (b); D is the optical density (a) and size (b) of fat bodies prepared by DOPC and DOPE in different proportions; E is the optical density (a) of fat bodies prepared by different ratios of TAG and CO Size (b); F is the optical density (a) and size (b) of the fat body incubated for 7 days at 4 ° C or room temperature.
  • a and C are optical microscopes; B and D are fluorescence microscopes.
  • FIG. 5 shows the isolation and purification of SMT3-Perilipin-2 protein.
  • A was separated by SDS-PAGE and analyzed by silver staining (top panel) and Western blot (bottom panel) for whole cell lysates before and after IPTG induction.
  • the black arrow points to the band corresponding to the SMT3-Perilipin-2 protein;
  • B is the result of SDS-PAGE and colloid blue staining of each component purified by the nickel ion metal affinity chromatography column.
  • C is a further purification of the SMT3-Perilipin-2 protein eluted from the nickel column by molecular sieves.
  • D is the SMT3-Perilipin-2 protein purified by SDS-PAGE analysis of nickel column (imidazole elution) And the components obtained from the molecular sieve.
  • Figure 6 shows that artificially reconstituted lipid droplet intrinsic/structural proteins from different species are recruited onto fat bodies.
  • Figure 7 shows the construction of artificial lipid droplets by recruiting the intrinsic/structural proteins of lipid droplets.
  • A is a schematic diagram of artificial lipid droplets;
  • B is a fat body that can recruit lipid droplet intrinsic/structural protein GFP fusion protein;
  • C is SMT3-Perilipin-2 protein saturation determination.
  • Figure 9 shows the results of silver staining analysis of SMT3-ATGL protein after incubation with fat bodies at room temperature or 4 °C.
  • Figure 10 shows the recruitment of ATGL and Apo A-I on fat bodies.
  • A is SMT3-ATGL protein saturation determination, wherein the lanes 1 to 7 correspond to the added SMT3-ATGL protein concentrations: 0.091, 0.132, 0.171, 0.209, 0.244, 0.278, 0.310 ⁇ g / microliter;
  • B is through the recruitment of high density
  • the lipoprotein apolipoprotein Apo AI constructs an artificial lipoprotein.
  • Figure 11 shows the preparation of drug-loaded fat bodies using vortexing and two-step centrifugation.
  • A is a flow chart for preparing a drug-loaded fat body: a is a component for preparing a drug-loaded fat body; b is a flow chart for preparing a drug-loaded fat body; B is a prepared drug-loaded fat body; C is determined by dynamic light scattering (DLS) The size of the drug-loaded fat body; D is the result of high performance liquid chromatography.
  • DLS dynamic light scattering
  • the adiposome described in the present invention is defined as a synthetic structure in which a natural or artificially synthesized neutral lipid is used as a core, a natural or artificial polar lipid monolayer film is wrapped.
  • the lipid composition and morphological structure of fat bodies are similar to those of lipid droplets, so it can be used as the main raw material for the preparation of artificial lipid droplets.
  • 1,2-Di-(9Z-octadecenyl)-sn-glycero-3-phosphocholine is a product of Avanti, and the product state is DOPC solution, that is, DOPC is dissolved in chloroform.
  • DOPC 1,2-Di-(9Z-octadecenyl)-sn-glycerol-3-phosphoethanolamine
  • DOPE DOPE
  • 1,2-Di-octadecanoyl-sn-glycero-3-phosphocholine is a product of Avanti, and the product state is DSPC solution, that is, DSPC is dissolved in chloroform.
  • Cholesterol oleate (CO) is a product of Alfa Aesar.
  • Nile Red is a product of Sigma-Aldrich.
  • LipidTOX red is a product of Invitrogen with catalog number H34476.
  • Anti-Perilipin-2 is Abcam's product catalog number ab108323.
  • Percoll is a GE Healthcare product.
  • the uranium acetate, 25% glutaraldehyde and EMbed 812 embedding kits are all products of Electron Microscopy Sciences (Hartfield, USA).
  • Osmium tetroxide (electron mirror grade) is a product of NAKALAI Tesque (Kyoto, Japan).
  • Tannin is a product of Sigma.
  • C57BL/6 mice are products of Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Dynamic Light Scattering Apparatus (DLS, Delsa Nano C Particle Analyzer) is a product of Beckman.
  • Vortex in the following examples was performed using a Vortex-Genie 1 vortexer (Scientific Industries, Inc., USA), and the "colloidal blue staining” was performed using a colloid blue staining kit (product of Invitrogen, product). The catalog number is LC6025).
  • Mitochondria were isolated from brown adipose tissue of C57BL/6 mice.
  • glutaraldehyde solution preparation method 25% glutaraldehyde is dissolved in pH 7.4, 0.1 M PB buffer, so that the mass concentration of glutaraldehyde in the system is 2.5%.
  • the solute of buffer B and its concentration in buffer are: 20 mM HEPES (15 mM - 25 mM HEPES in practical application), 100 mM KCl (80 mM - 120 mM KCl in practical use), 2 mM MgCl 2 (1.5 in practical application) Available -2.5mM MgCl 2); the solvent is deionized water; the pH was 7.4 (7.2-7.6 Available practical application).
  • the liposome was mixed with phosphatidylcholine and phosphatidylethanolamine at a mass ratio of 3:7, and then prepared by using a single-layer liposome prepared by a polycarbonate filter micro-extruder (product of Avanti) having a pore size of 100 nm.
  • Methods refer to Nakatogawa, H., Ichimura, Y. & Ohsumi, Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165-178, doi: 10.1016/j.cell.2007.05 .021 (2007).
  • the fat body or lipid droplets were observed by fluorescence microscopy.
  • the specific steps were as follows: the fat body or lipid droplets were diluted 1:10 times with Nile Red (concentration of 1 ⁇ g/ml) or LipidTOX red (LipidTOX red with buffer B). Dilute and obtain LipidTOX red dilution), incubate for 30 min at room temperature, take 6 ⁇ l of the fat body or lipid droplets after incubation, add dropwise to the glass slide, and then add 2 ⁇ l anti-fluorescence quencher (Biyuntian company product, catalog number is P0126) Mix and cover the coverslip. Fluorescence images were obtained using a Zeiss M2 fluorescence microscope, an Olympus FV1000 fluorescence confocal microscope or a DeltaVision OMX V3 super-resolution microscope.
  • the fat body or mitochondria were observed by fluoroscopy.
  • the specific steps were as follows: The fat body or mitochondria were quickly mixed with the melted 3% low melting point agarose, and solidified on ice, and then cut into small pieces of about 1 mm 3 .
  • the pellet was fixed in a 2.5% glutaraldehyde solution for 30 min and then fixed with 1% osmium tetroxide dissolved in pH 7.4, 0.1 M PB buffer for 1 h at room temperature to obtain a fixed block.
  • the fixed block was washed with deionized water, dehydrated with ethanol, then infiltrated and embedded with an EMbed 812 embedding kit, and polymerized at 60 ° C for 24 h, and a 70 nm ultrathin section was prepared using a Leica EM UC6 ultramicrotome. Micrographs were obtained at 100 kV using a Tecnai Spirit (FEI) transmission electron microscope.
  • FEI Tecnai Spirit
  • the fat body or mitochondria were observed by cryo-electron microscopy.
  • the specific steps were as follows: 4 ⁇ l of fat body or mitochondria were taken and dropped on a copper grid. It was blotted with a Vitrobot Mark IV (product of FEI Corporation) at 100% humidity for 3 seconds, and then rapidly immersed in liquid ethane previously cooled with liquid nitrogen to vitrify. Micrographs were taken at 300 kV using a Titan Krios cryo-electron microscope (product of FEI) equipped with a Gatan UltraScan 4000 camera (model 895).
  • Buffer T was pH 7.4, buffer containing 50 mM Tris-HCl and 150 mM NaCl.
  • the vector pET28a is a product of Novagen.
  • the vector pGEX-6p-1 was purchased from Novagen.
  • the nickel ion metal affinity chromatography column packing is a Chelating Sepharose Fast Flow chelated with nickel ions, which is a product of Amersham Biosciences.
  • the GST affinity chromatography column packing is a product of Bio-Bio Engineering (Shanghai) Co., Ltd.
  • Modified pET28a plasmid is described in Hu, W., Wu, H., Zhang, H., Gong, W. & Perrett, S. Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces Cerevisiae. Biomol NMR Assign 9, 329-332, doi: 10.1007/s 12104-015-9603-5 (2015).
  • the "transformed pET28a plasmid” is hereinafter named the vector pET28a-SMT3.
  • step (1) After completing step (1), add 100 ⁇ l of buffer B and 5 mg of TAG to a microcentrifuge tube, vortex for 4 min (vortex for 10 s, stop for 10 s) to obtain a milky white lipid mixture 1 (ie, the initial preparation component).
  • the lipid mixture 1 was centrifuged at 20000 g for 5 min (in practical applications, 18000-22000 g was centrifuged for 3-7 min). After centrifugation, the bottom of the microcentrifuge tube is the precipitate component 1, and the liquid phase system exhibits two layers (the upper layer is white band 1, and the white band 1 or less is solution 1).
  • step (2) After the completion of the step (2), the solution 1 and the precipitate component 1 are discarded by extraction, the white band 1 is retained, 100 ⁇ l of the buffer B is added, and the mixture is vortexed to obtain a milky white lipid mixture 2, and the lipid is obtained.
  • Mixture 2 was centrifuged at 20,000 g for 5 min (in practical applications, 18000-22000 g was centrifuged for 3-7 min). After centrifugation, if there is a precipitate at the bottom of the microcentrifuge tube, which is the precipitate component 2, the liquid phase system exhibits two layers (the upper layer is white band 2, and the white band 2 or less is solution 2).
  • Step (4) is the repetition of step (3), and the number of repetitions of step (3) in the actual application is based on the absence of precipitation of the white layer under the layer.
  • step (4) After completing step (4), take white band 3, add 100 ⁇ l of buffer B, vortex and mix, centrifuge at 1000 g for 5 min (800-1200 g for 3-7 min in actual application), after centrifugation, the liquid phase system presents two Layer layering (the upper layer is white strip 4, and the white strip 4 is below solution 4). The solution 4 is collected, which is the fat body a.
  • the white strip 4 in the above step is named as the upper layer component.
  • the precipitate component 1 and the precipitation component 2 in the above steps were mixed and designated as a precipitation component.
  • the precipitate component and the upper component in the above step were mixed and designated as a mixed component.
  • FIG. 1A The specific process for preparing fat body a by the above method is shown in Figure 1A (where a is the preparation of fat body a
  • the component, b is a flow chart for preparing fat body a).
  • the morphology of the components in the process of preparing fat body a was observed by optical microscopy and fluorescence microscopy, and the TEM was positively stained with a microscope.
  • the results were as follows: the initial preparation component, the precipitation component and the upper component were neutralized with neutral lipids.
  • the globular structure there are many other types of structures (B in Figure 1); almost all of the structure 4 are spherical, and all are Nile Red positive, and the size of the Nile Red labeled structure and the optical microscope image The size of the globular structure is consistent, which indicates that these globular structures contain the TAG core (C in Figure 1); the fat body a and the liposome are directly stained by transmission electron microscopy, and the results show that there is a significant difference between the two (Figure 1 D).
  • the average size of fat body a was measured by dynamic light scattering instrument to be about 189 nm (B in Fig. 2), and then the literature (Ding Y, Zhang S, et al. Isolating lipid droplets from multiple species. Nat Protoc. 2013) was used. Jan;8(1):43-51.doi:10.1038/nprot.2012.142.Epub 2012 Dec 6.) Calculate the DOPC and total lipids (total lipids ie TAG and DOPC) in fat body a The volume ratio is about 6.6%.
  • the extract is a fat body a, a lipid mixture 1 (i.e., an initial preparation component) or a mixed component.
  • step b the total lipid obtained in step a was dissolved in 100 ⁇ l of chloroform, and 10 ⁇ l was applied to a silica gel plate;
  • step b after completion of step b, the sample is developed in a developing solvent of n-hexane: diethyl ether: glacial acetic acid (80:20:1 by volume) to separate TAG;
  • step c the organic solvent (ie, n-hexane, diethyl ether and glacial acetic acid in step c) is evaporated in air, and the silica gel plate is again in chloroform:methanol:glacial acetic acid:water (volume ratio: 75:13: 9:3) solvent system unfolded to separate DOPC;
  • organic solvent ie, n-hexane, diethyl ether and glacial acetic acid in step c
  • step d stain with saturated iodine vapor and quantify the gray scale of the lipid spots using ImageJ software.
  • Lipid droplets were isolated from the bacteria RHA1 and named as liver tissue-lipid (Liver LD), brown adipose tissue-lipid (BAT LD) and RHA1-lipid (RHA1 LD).
  • Liver LD liver tissue-lipid
  • BAT LD brown adipose tissue-lipid
  • RHA1 LD RHA1-lipid
  • Fat body a, RHA1 LD, Liver LD and BAT LD are all milky solutions.
  • the average size of fat body a, RHA1 LD, Liver LD and BAT LD was measured by dynamic light scattering instrument.
  • BAT LD had the largest particle size and the average diameter was 1848 nm;
  • RHA1 LD had the smallest particle size and the average diameter was 493 nm;
  • fat body a The average diameter is 189 nm (B in Figure 2).
  • the above results are basically consistent with those obtained by an optical microscope (D in Fig. 2).
  • Fat bodies or lipid droplets were observed by fluorescence microscopy.
  • Fat bodies a, RHA1 LD, Liver LD, and BAT LD all exhibited a globular structure (D in Figure 2), indicating that they all contained a TAG core.
  • step 2 To optimize the conditions for the preparation of fat bodies, we systematically changed the two main factors in the vortex and two-step method described in step 1, namely the vortex time in step (2) and the two raw materials (phospholipids and neutral lipids).
  • the ratio of the mass is measured by measuring the optical density (OD 600 ) at a wavelength of 600 nm to reflect the yield of the fat body, and the size of the fat body is measured by a dynamic light scattering instrument.
  • step 1 the vortex of (1) in step 1 is replaced by vortex 1 min, vortex 2 min and vortex 3 min, respectively, and the other steps are unchanged, and fat body b, fat body c and fat body are obtained.
  • d then measure the size and optical density of fat body a, fat body b, fat body c or fat body d, respectively.
  • the phospholipid in the preparation of fat body a is specifically DOPC, the neutral lipid is specifically TAG, and the ratio of DOPC: TAG is 2:5.
  • step 1 replace 2 mg of DOPC of (1) in step 1 with 0.25 mg of DOPC, 0.5 mg of DOPC, 1 mg of DOPC and 3 mg of DOPC, respectively, and the other steps are unchanged to obtain fat body a1 (DOPC: TAG The ratio is 1:20), fat body a2 (DOPC: TAG ratio is 1:10), fat body a3 (DOPC: TAG ratio is 1:5) and fat body a4 (DOPC: TAG ratio is 3:5) Then, the size and optical density of the fat body a1, the fat body a2, the fat body a3, and the fat body a4 were measured, respectively.
  • step 2 According to the method of the above step 1, replace 2 mg DOPC of (1) in step 1 with 1.33 mg DOPC and 0.67 mg DSPC, 1 mg DOPC and 1 mg DSPC, 0.67 mg DOPC and 1.33 mg DSPC, and 2 mg DSPC, respectively. , get fat body A1 (DOPC: DSPC ratio is 2:1), fat Fat body A2 (DOPC: DSPC ratio is 1:1), fat body A3 (DOPC: DSPC ratio is 1:2) and fat body A4 (phospholipid is only DSPC), then measure fat body A1, fat body A2 The size and optical density of fat body A3 and fat body A4.
  • the 2 mg DOPC of (1) in step 1 is replaced by 1.33 mg DOPC and 0.67 mg DOPE, 1 mg DOPC and 1 mg DOPE, 0.67 mg DOPC and 1.33 mg DOPE, and 2 mg DOPE, respectively, and the other steps are unchanged.
  • Obtained fat body B1 DOPC: DOPE ratio of 2:1
  • fat body B2 DOPC: DOPE ratio of 1:1
  • fat body B3 DOPC: DOPE ratio of 1:2
  • fat body B4 The phospholipid was only DOPE), and then the size and optical density of the fat body B1, the fat body B2, the fat body B3, and the fat body B4 were measured, respectively.
  • step 4 replace 5 mg TAG of (1) in step 1 with 4.17 mg TAG and 0.83 mg CO, 4 mg TAG and 1 mg CO, 3.75 mg TAG and 1.25 mg CO, and 3.33 mg TAG and 1.67 mg CO, respectively.
  • the steps were unchanged, and the fat body C1 (the ratio of TAG:CO was 5:1), the fat body C2 (the ratio of TAG:CO was 4:1), and the fat body C3 (the ratio of TAG:CO was 3:1) were obtained.
  • the fat body C4 (TAG:CO ratio is 2:1), and then the size and optical density of the fat body C1, the fat body C2, the fat body C3, and the fat body C4 were measured, respectively.
  • the fat body a was incubated at room temperature or 4 ° C and incubated for 7 days.
  • the size of the fat body a, the optical density at OD 600 , and the fat body were observed by optical microscopy and fluorescence microscopy throughout the incubation period.
  • Example 2 Recombinant artificial lipid droplets by recruitment of innate proteins and/or functional proteins and recombinant lipoprotein recombinants by recruitment of apolipoproteins
  • Lipid droplets are highly dynamic and participate in a wide variety of physiological functions and interactions through the mediation of multiple lipoproteins. Proteins on lipid droplets can be broadly classified into two categories: intrinsic proteins and dynamic proteins. Intrinsic proteins are thought to be selectively localized on lipid droplets, involved in the control of lipid droplet size, and mediate the localization of dynamic proteins. For example, phosphorylated PLIN1 causes hormone-sensitive lipase (HSL) to localize on lipid droplets. Therefore, in the use of fat bodies to deconstruct the function of lipid droplets, it is necessary to load the fat droplet intrinsic protein on the fat body, and then further recruit functional proteins based on the successful recruitment of the intrinsic protein to produce artificial lipid droplets.
  • HSL hormone-sensitive lipase
  • the intrinsic protein of lipid droplets is specifically an intrinsic protein of lipid droplets derived from different organisms from bacteria to mammals, including MLDS protein on RHA1-lipid droplets (RHA1LD) isolated from the oleaginous fungus RHA1, nematode lipid
  • RHA1LD RHA1-lipid droplets isolated from the oleaginous fungus RHA1, nematode lipid
  • the MDT-28 protein and the Perilipin-2 protein in mammalian non-fat cells the functional protein is ATGL.
  • the apolipoprotein is specifically apolipoprotein A1 (Apo A-I) derived from human.
  • the DNA molecule shown in SEQ ID NO: 1 in the sequence listing is synthesized (the MLDS gene), and the DNA molecule shown in SEQ ID NO: 1 is the protein shown in SEQ ID NO: 2 (i.e., MLDS protein, NCBI number WP_005261062.1).
  • Synthesize the DNA molecule shown in SEQ ID NO: 3 in the sequence listing ie, MDT-28 gene, NCBI number NM_001129054.2
  • the DNA molecule shown in SEQ ID NO: 3 encodes the protein shown in SEQ ID NO: 4 (ie, MDT-28 protein, NCBI number) Is NP_001122526.1).
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence listing (i.e., the Perilipin-2 gene) is synthesized, and the DNA molecule shown in SEQ ID NO: 5 encodes the protein shown in SEQ ID NO: 6 (i.e., Perilipin-2 protein, NCBI number NP_001113.2).
  • the DNA molecule shown in SEQ ID NO: 7 in the sequence listing (i.e., the ATGL gene) is synthesized, and the DNA molecule shown in SEQ ID NO: 7 encodes the protein shown in SEQ ID NO: 8 (ie, ATGL, NCBI number NP_065109.1).
  • the DNA molecule shown in SEQ ID NO: 9 in the sequence listing (i.e., MLDS-GFP fusion gene) is synthesized, and the DNA molecule shown in SEQ ID NO: 9 encodes the protein shown in SEQ ID NO: 10 (i.e., MLDS-GFP fusion protein).
  • the DNA molecule shown in SEQ ID NO: 11 in the sequence listing (i.e., the MDT-28-GFP fusion gene) is synthesized, and the DNA molecule shown in SEQ ID NO: 11 encodes the protein shown in SEQ ID NO: 12 (i.e., MDT-28-GFP fusion protein).
  • the DNA molecule shown in SEQ ID NO: 13 in the sequence listing (i.e., the Perilipin-2-GFP fusion gene) is synthesized, and the DNA molecule shown in SEQ ID NO: 13 encodes the protein shown in SEQ ID NO: 14 (i.e., Perilipin-2-GFP fusion protein).
  • Bovine serum albumin was purchased from Sigma, catalog number A4612.
  • vector pET28a-SMT3 Replacing the fragment between the restriction endonucleases EcoRI and XhoI of the vector pET28a-SMT3 by the DNA molecule shown in SEQ ID NO: 5 in the sequence listing (vector pET28a-SMT3 is cleaved by restriction endonucleases EcoRI and XhoI)
  • a large fragment and a small fragment, and a large fragment is ligated to the DNA molecule shown in SEQ ID NO: 5 to obtain a recombinant plasmid.
  • the recombinant plasmid expresses a soluble protein having a 6-His tag and an SMT3 domain fused at the N-terminus.
  • the recombinant plasmid constructed in the step (1) was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant Escherichia coli containing the recombinant plasmid.
  • the recombinant Escherichia coli obtained in the step (2) is inoculated into 2 ⁇ YT medium (16 g of peptone, 10 g of yeast extract and 4 g of sodium chloride dissolved in 1000 mL of distilled water), and cultured at 37° C., when the recombinant Escherichia coli is cultured.
  • the OD 600 of the bacterial solution reached 0.6 (the culture broth was named as the pre-induction bacterial liquid), and isopropyl ⁇ -D-1-thiogalactoside (IPTG) was added to make the concentration of IPTG in the system 0.4 mM. It was induced at 16 ° C for 24 h (the culture broth after IPTG induction was named as the induced bacterial liquid).
  • the cells of the pre-bacterial or induced bacterial cells were collected (4000 rpm, centrifuged for 20 min), resuspended in buffer T, and then lysed with a high-pressure cell disrupter (model JNBIO JN-3000 PLUS) to obtain whole cell lysis. liquid.
  • Whole cell lysates were subjected to SDS-PAGE and then analyzed by silver staining and Western blotting. The results showed (A in Figure 5, where a is the result of silver staining analysis, b is the result of Western blot analysis, the arrow points to the band corresponding to SMT3-Perilipin-2 protein), and a large amount of SMT3-Perilipin-2 protein in the bacterial solution after induction expression.
  • step (3) Take the whole cell lysate of the cells of the bacterial liquid after the induction of step (3), centrifuge at 30000 g for 50 min, and obtain the supernatant and precipitate, and apply the supernatant to the nickel ion metal affinity chromatography column (the filler is chelated) Chelating Sepharose Fast Flow with nickel ion, the cartridge was purchased from Thermo, catalog number 29924, column volume 4 mL, inner diameter 1.2 cm), and then 12 column volumes were eluted with buffer T containing 20 mM imidazole to remove non- The target protein was eluted once with 3 volumes of buffer T containing 500 mM imidazole to collect the target protein twice (all buffers containing the target protein after the column was passed), and then centrifuged ultrafiltration tube (Amicon) The imidazole was removed by buffer exchange, and purified again by Size Exclusion Chromatography (SEC) (HiLoad 16/600 Superdex 200 column, GE product), the
  • component 1 retention volume 45mL-57mL
  • component 2 retention volume 57mL-67mL
  • component 3 retention volume 67mL-79mL
  • component 4 restored volume It is 79 mL-94 mL).
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence table of (1) in step 1 is replaced with the DNA molecule shown in SEQ ID NO: 1 in the sequence listing, and the vector pET28a-SMT3 is replaced with the vector pGEX-6p-1, which is restricted.
  • the endonuclease XhoI was replaced by BamHI, and the nickel ion metal affinity column of step (4) was replaced with a GST affinity chromatography column, and the imidazole was replaced with reduced glutathione.
  • the other steps were unchanged and purified.
  • MLDS protein The endonuclease XhoI was replaced by BamHI, and the nickel ion metal affinity column of step (4) was replaced with a GST affinity chromatography column, and the imidazole was replaced with reduced glutathione. The other steps were unchanged and purified. MLDS protein.
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence listing of (1) in step 1 is replaced with The DNA molecule shown in SEQ ID NO: 3 in the sequence listing, the vector pET28a-SMT3 was replaced with the vector pGEX-6p-1, the restriction enzymes EcoRI and XhoI were replaced with the restriction enzymes BamHI and NotI, step (4)
  • the nickel ion metal affinity chromatography column was replaced by a GST affinity chromatography column, and the imidazole was replaced with reduced glutathione. The other steps were unchanged, and the purified MDT-28 protein was obtained.
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence table of (1) in step 1 is replaced with the DNA molecule shown in SEQ ID NO: 9 in the sequence listing, and the vector pET28a-SMT3 is replaced with the vector pET28a, and the other steps are unchanged.
  • a purified MLDS-GFP fusion protein was obtained.
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence table of (1) in step 1 is replaced with the DNA molecule shown in SEQ ID NO: 11 in the sequence listing, and the vector pET28a-SMT3 is replaced with the vector pET28a and restriction endonuclease EcoRI.
  • the restriction enzyme BamHI was replaced with the other steps, and the purified MDT-28-GFP fusion protein was obtained.
  • the DNA molecule shown in SEQ ID NO: 5 in the sequence table of (1) in step 1 is replaced with the DNA molecule shown in SEQ ID NO: 13 in the sequence listing, and the vector pET28a-SMT3 is replaced with the vector pET28a, and the other steps are unchanged.
  • a purified Perilipin-2-GFP fusion protein was obtained.
  • the DNA molecule shown by the sequence 5 in the sequence table of (1) in the step 1 is replaced with the DNA molecule shown by the sequence 7 in the sequence table, and the Escherichia coli BL21 (DE3) of the step (2) is replaced with E. coli Rosetta, the other steps were unchanged, and the purified SMT3-ATGL protein was obtained.
  • the purified Apo A-I was obtained by the method described in Peitsch, M.C. et al. A purification method for apolipoprotein A-I and A-II. Anal Biochem 178, 301-305 (1989).
  • A1 5 ⁇ g of SMT3-Perilipin-2 protein, 25 ng of Ulp1 and 50 ⁇ l of the fat body a prepared in Example 1 were mixed to obtain a mixed system of 100 ⁇ l.
  • step a1 After completing step a1, incubate for 1 h at room temperature (in practice, on ice) to obtain a mixed system.
  • step a2 After completion of step a2, the mixed system is taken, 20,000 g, centrifuged for 5 min, and the liquid phase system presents two layers (the upper layer is a fat body), and the lower layer solution is removed, and the upper layer is retained.
  • step a3 the upper layer of step a3 is completed, resuspended in 100 ⁇ l of buffer B, 20000 g, centrifuged for 5 min, the liquid phase system presents two layers (the upper layer is a fat body), the lower layer solution is removed, and the upper layer is retained.
  • step a4 The upper layer of step a4 is completed, resuspended in 100 ⁇ l of buffer B, 20000 g, centrifuged for 5 min, and the liquid phase system is layered in two layers, the lower layer solution is removed, and the upper layer (ie, fat body) is retained.
  • the 5 ⁇ g SMT3-Perilipin-2 protein, the fat body obtained in the step a5, and the lower layer solution extracted in the step a3 were all subjected to silver staining after SDS-PAGE.
  • lane 7 is total protein (SMT3-Perilipin-2 protein)
  • lane 8 is liposome-collected protein (step a5 to obtain fat body)
  • lane 9 is solution protein (step A3 pumped lower layer solution)).
  • the results showed that about 50% of the Perilipin-2 protein was recruited to the fat body a.
  • the fat body recruited with Perilipin-2 protein was named as artificial lipid droplet Perilipin-2.
  • SMT3-Perilipin-2 protein 25 ng Ulp1 and 30 ⁇ l of fat body a prepared in Example 1 were mixed to obtain a mixed system of 50 ⁇ l.
  • the concentration of SMT3-Perilipin-2 protein in the mixed system was 0.015 ⁇ g/ ⁇ l. 0.030 ⁇ g/ ⁇ l, 0.056 ⁇ g/ ⁇ l, 0.080 ⁇ g/ ⁇ l, 0.101 ⁇ g/ ⁇ l, 0.137 ⁇ g/ ⁇ l, 0.166 ⁇ g/ ⁇ l or 0.191 ⁇ g/ ⁇ l.
  • step b2 after completion of step b1, incubate for 1 h at room temperature (actually on ice) to obtain a mixed system.
  • step b2 the mixed system is taken, 20,000 g, centrifuged for 5 min, and the liquid phase system presents two layers (the upper layer is a fat body), and the lower layer solution is removed, and the upper layer is retained.
  • step b4 taking the upper layer of step b3, resuspending in 100 ⁇ l of buffer B, 20000 g, centrifugation for 5 min, the liquid phase system presents two layers (the upper layer is a fat body), the lower layer solution is removed, and the upper layer is retained.
  • step b4 taking the upper layer of step b4, resuspending in 100 ⁇ l of buffer B, 20000 g, centrifugation for 5 min, the liquid phase system presents two layers, the lower layer solution is removed, and the upper layer (ie, fat body) is retained.
  • the 5 ⁇ g MLDS protein, the fat body obtained in step b5, and the lower layer solution extracted in step b3 were all subjected to silver staining after SDS-PAGE.
  • lane 1 is the total protein (MLDS protein)
  • lane 2 is the liposome-collected protein (step b5 to obtain the fat body)
  • lane 3 is the solution protein (step b3) Lower layer solution)).
  • the results showed that about 80% of the MLDS protein was recruited to the fat body a.
  • the fat body recruited with the MLDS protein was named artificial lipid droplet MLDS.
  • the MLDS protein in step (2) was replaced with the MDT-28 protein, and the other steps were unchanged.
  • the results are shown in lanes 4, 5 and 6 of Figure 6 (lane 4 is total protein (MDT-28 protein), lane 5 is liposome-collecting protein, and lane 6 is solution protein).
  • the results showed that about 90% of the MDT-28 protein was recruited to the fat body a.
  • the fat body recruited with MDT-28 protein was named as artificial lipid droplet MDT-28.
  • the MLDS protein in step (2) was replaced with bovine serum albumin, and the other steps were unchanged.
  • the results are shown in lanes 11, 12 and 13 of Figure 6 (lane 11 is total protein (bovine serum albumin), lane 12 is liposome-collected protein, and lane 13 is solution protein). The results showed that bovine serum albumin was not recruited to fat body a.
  • the fat body recruited with protein is named as an artificial lipid droplet.
  • step 1 (1) 5 ⁇ g of SMT3-Perilipin-2 protein was replaced with 10 ⁇ g of purified Perilipin-2-GFP fusion protein, and the other steps were unchanged, and artificial lipid droplet Perilipin-2-GFP was obtained.
  • the (5) 5 ⁇ g MLDS protein in step 1 was replaced with 10 ⁇ g of the purified MLDS-GFP fusion protein to obtain an artificial lipid droplet MLDS-GFP.
  • 5 ⁇ g of MDT-28 protein in step 1 was replaced with 10 ⁇ g of MDT-28-GFP fusion protein to obtain artificial lipid droplet MDT-28-GFP.
  • step 1 SMT3-Perilipin-2 protein was replaced with SMT3-ATGL protein, 50 ⁇ l of fat body a was replaced with 30 ⁇ l of fat body a, and the other steps were unchanged, and a fat body recruited with ATGL was obtained.
  • the results are shown in Figure 9.
  • the left panel shows the results of incubation at room temperature.
  • the right panel shows the results of incubation at 4 °C.
  • Lanes 1 and 5 are Marker
  • lanes 2 and 6 are total proteins (SMT3-ATGL protein)
  • lanes 3 and 7. Proteins are recruited for liposomes
  • lanes 4 and 8 are solution proteins).
  • the results showed that about 1/3 of ATGL was recruited to fat body a.
  • the fat body recruited with ATGL was named as artificial lipid drop ATGL.
  • SMT3-ATGL protein was mixed with 50 ⁇ l of fat body a prepared in Example 1 to obtain a mixed system of 100 ⁇ l.
  • the concentration of SMT3-ATGL protein in the mixed system was 0.091 ⁇ g/ ⁇ l, 0.132 ⁇ g/ ⁇ l, 0.171. ⁇ g/ ⁇ l, 0.209 ⁇ g/ ⁇ l, 0.244 ⁇ g/ ⁇ l, 0.278 ⁇ g/ ⁇ l or 0.310 ⁇ g/ ⁇ l.
  • an artificial lipid droplet was obtained, subjected to SDS-PAGE, and then silver stained, or subjected to Western blot detection using an ATGL antibody (Cell Signaling Technology, catalog number 2138) as a primary antibody.
  • ATGL antibody Cell Signaling Technology, catalog number 2138
  • the results are shown in Figure 10, A (where a is the result of silver staining and b is the result of Western blot). The results showed that when the concentration of ATGL in the reaction system was 0.209 ⁇ g/ ⁇ l, the recruitment of fat body a to ATGL was saturated.
  • the (5) 5 ⁇ g MLDS protein in step 1 was replaced with 9 ⁇ g Apo A-I, and the other steps were unchanged, and a fat body recruited with Apo A-I was obtained.
  • the results are shown in Figure 10, B (lane 1 is a liposome-collected protein, lane 2 is a solution protein, and lane 3 is a total protein (Apo A-I)).
  • the results showed that about 50% of Apo A-I was recruited to fat body a.
  • the fat body recruited with Apo A-I was named as artificial lipoprotein Apo A-I.
  • the fat body a' was prepared in the same manner as in the first step of Example 1, except that the commercially available glycerol trioleate was used in place of the triglyceride in Example 1, and the fat body a' was obtained in the step (5).
  • Each of the fat body a' was subjected to the tests of Example 2, and the results were basically the same as those of the fat body a, and there was no significant difference.
  • triolein available from Sigma, catalog number 92860.
  • Example 3 application of fat body as a drug carrier
  • the drug in this embodiment is a compound represented by formula a;
  • the drug is a fat-soluble drug that is miscible with triglycerides and is a drug candidate for the treatment of type 2 diabetes and/or metabolic disorders, specifically a product of the TOCRIS company catalog number 5869, under the trade name CDN 1163.
  • the ZORBAX SB-C18 column is a product of Agilent.
  • TAG solution 10 mg was added to a microcentrifuge tube, 100 ⁇ l of chloroform was added thereto, and fully dissolved to obtain a TAG solution.
  • step (5) After the completion of the step (5), the solution A and the precipitate component A are discarded by extraction, the white band is retained, 100 ⁇ l of the buffer B is added, and the mixture is vortexed to obtain a milky white lipid mixture B, and the lipid is obtained.
  • Mixture B is centrifuged at 20,000 g for 5 min (in practical applications, 18000-22000 g can be centrifuged for 3-7 min). After centrifugation, if there is a precipitate at the bottom of the microcentrifuge tube, it is the precipitation component B, and the liquid phase system presents two layers (the upper layer is white with B, and the white layer with B below is solution B).
  • the solution B and the precipitate component B are discarded by extraction, the white band B is retained, 100 ⁇ l of the buffer B is added, and the mixture is vortexed to obtain a milky white lipid mixture C, and the lipid is obtained.
  • the mixture C was centrifuged at 20000 g for 5 min (in practical applications, 18000-22000 g was centrifuged for 3-7 min). After centrifugation, the liquid phase system exhibited two layers (the upper layer was white with C and the white with C was below solution C).
  • Step (7) is the repetition of step (6), and the number of repetitions of step (6) in the actual application is white. There is no sediment in the lower layer.
  • step (7) After completion of step (7), take white with C, add 100 ⁇ l of buffer B, vortex and mix, centrifuge at 1000g for 5min (800-1200g for 3-7min in actual application), after centrifugation, the liquid phase system presents two The layers are layered (the upper layer is white with diced, and the white layer with diced portion is solution). The solution solution is collected, which is a drug-loaded fat body.
  • FIG. 11A The specific procedure for preparing the drug-loaded fat body by the above method is shown in Figure 11A (wherein a is a component for preparing a drug-loaded fat body, and b is a flow chart for preparing a drug-loaded fat body).
  • the drug-loaded fat body prepared by the above method is shown in FIG. 11B (left is the fat body a prepared in the first step of the first embodiment, and the right is the drug-loaded fat body).
  • the average size of the fat body a was measured by a dynamic light scattering instrument to be about 166.1 nm (C in Fig. 11).
  • CDN 1163 10 mg was accurately weighed into a 100 ml volumetric flask, dissolved in anhydrous methanol (chromatographic grade) and made up to 100 ml, and formulated into 100 ⁇ g/ml CDN 1163 mother liquor. Then, it was further diluted with anhydrous methanol (chromatographic grade) to obtain a standard solution having a concentration of 20 and 4 ⁇ g/mL, respectively. Peak areas of different concentrations of CDN 1163 were determined using an Agilent 1260 liquid chromatograph equipped with a ZORBAX SB-C18 column (5.0 ⁇ m, 4.6 mm x 150 mm), 3 replicates. The mobile phase consisted of methanol and water with a volume ratio of methanol to water of 85:15 and a flow rate of 1.0 mL/min. The detection wavelength was 245 nm.
  • the standard curve is drawn by taking the concentration of CDN1163 as the abscissa and the peak area as the ordinate.
  • the linear relationship of CDN 1163 concentration in 0-100 ⁇ g/ml was good.
  • Example 1 The above results indicate that the fat body prepared in Example 1 can be used as a pharmaceutical carrier.
  • the fat body, artificial lipid droplets and artificial lipoprotein prepared by the method disclosed by the invention can be used as a drug carrier, thereby accomplishing various biological and medical goals, and have important application value.

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Abstract

一种脂肪体的制备方法及其应用。具体提供一种制备由中性脂质和单层磷脂膜组成的脂肪体的方法，包括下述步骤：a1)将磷脂和中性脂质在缓冲液中进行涡旋以实现两者的反应，然后进行离心，收集上层液相；a2)将步骤a1)得到的上层液相进行两次以上的纯化；每次纯化的方法为：将上层液相与所述缓冲液混匀，然后使其分层，收集上层液相；a3)将步骤a2)得到的上层液相与所述缓冲液混合，然后使其分层，收集下层液相，其中含有脂肪体。所述方法制备的脂肪体，可招募一种或几种固有蛋白和/或功能蛋白、获得人工脂滴，可招募一种或几种载脂蛋白、获得人工脂蛋白，均在制备药物和/或药物载体中具有重要作用。

Description

一种脂肪体的制备方法及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种脂肪体的制备方法及其应用。
背景技术
脂滴(lipid droplet,LD)是一种细胞器，它由中性脂质核心，单层磷脂膜与相关蛋白组成，这一细胞器的功能包括：1)脂质的合成，存储，代谢和运输；2)蛋白质的存储和降解；3)脂质信号分子和激素的生产和修饰；4)与其他细胞器的相互作用。由于脂滴具有上述的多种功能，许多人类的疾病，特别是代谢性疾病，与脂滴的形成和动态变化息息相关，然而，脂滴的形成机制和动态变化研究依然困难重重。目前仍然缺乏对脂滴如何形成、脂滴的功能及调控机制的全面了解。脂滴的组成的复杂性及其与其它细胞器的相互作用干扰了我们解构这些机制的能力。
在过去的十年中，人们致力于脂滴的分离纯化，并通过蛋白质组和脂质组研究阐明脂滴的蛋白质和脂质组成。可是蛋白质组和其他的组分研究发现，分离得到的脂滴几乎总是含有内质网、线粒体和其它细胞器的碎片。可见，制备结构和组成上都接近天然脂滴的人工脂滴(Artificial Lipid Droplets，ALDs)是非常必要的。
除脂滴以外，人体内还有一类由单层磷脂膜包裹中性脂，表面覆有蛋白的结构，称为脂蛋白(lipoprotein)。虽然脂蛋白与脂滴的结构非常类似，但二者定位不同，表面蛋白不同：脂滴存在于细胞中，而脂蛋白存在于血液中；脂滴上的主要蛋白为脂滴固有/结构蛋白(lipid droplet resident/structural protein)，而脂蛋白上的主要蛋白为载脂蛋白(apolipoprotein)。不同的脂蛋白表面有不同的载脂蛋白。脂蛋白的种类和比例对人体健康有着非常重要的影响。高密度脂蛋白(high-density lipoprotein，HDL)是胆固醇逆向运输(cholesterol reverse transport)的关键组分，可防止胆固醇在血管壁上的沉积，对动脉粥样硬化等心脑血管疾病有非常重要的预防作用。人工脂蛋白或可用于高密度脂蛋白的补充制剂或其他药物载体。
发明公开
本发明所要解决的问题是提供药物载体及一种体外研究脂滴的体系。
为解决上述问题，本发明首先提供了一种脂肪体的制备方法，所述脂肪体由中性脂质和单层磷脂膜组成。
本发明所提供的制备脂肪体的方法，包括下述步骤：a1)将磷脂和中性脂质在缓冲液中进行涡旋以实现两者的反应，然后进行离心，收集上层液相，从上层液相中分离得到脂肪体。
所述“从上层液相中分离得到脂肪体”可包括如下步骤：a2)将所述上层液相进行两次以上的纯化；每次纯化的方法可为：将上层液相与所述缓冲液混 匀，然后使其分层，收集上层液相；a3)将步骤a2)得到的上层液相与所述缓冲液混合，然后使其分层，收集下层液相，其中含有脂肪体。
所述缓冲液可为缓冲液B。
所述缓冲液B的溶质及其在缓冲液中浓度可为：15mM～25mM HEPES，80mM～120mM KCl，1.5～2.5mM的MgCl2；溶剂可为去离子水；pH可为7.2～7.6。
所述缓冲液B的溶质及其在缓冲液中浓度具体可为：20mM HEPES，100mM KCl，2mM的MgCl2；溶剂具体可为去离子水；pH具体可为7.4。
所述步骤a1)中，所述涡旋的参数可为：总时长为3～5min。所述步骤a1)中，所述离心的参数可为：18000～22000g、3～7min。所述步骤a1)中，所述涡旋的参数具体可为：总时长4min。所述步骤a1)中，所述离心的参数具体可为：20000g、5min。
所述步骤a2)中，所述“上层液相进行两次以上的纯化”中的次数以上层液相与所述缓冲液混匀，分层后无沉淀为准。所述步骤a2)中，所述“使其分层”是通过离心实现的，所述离心的参数可为：18000～22000g、3～7min。所述步骤a2)中，所述“使其分层”是通过离心实现的，所述离心的参数具体可为：20000g、5min。
所述步骤a3)中，所述“使其分层”是通过离心实现的，所述离心的参数可为：800～1200g、3～7min。所述步骤a3)中，所述“使其分层”是通过离心实现的，所述离心的参数具体可为：1000g、5min。
所述磷脂为b1)、b2)或b3)：b1)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱(DOPC)；b2)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱(DOPC)和1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸乙醇胺(DOPE)；b3)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱(DOPC)和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱(DSPC)。
所述中性脂质为c1)或c2)：c1)甘油三酯；c2)胆固醇油酸酯(cholesteryloleate,CO)和甘油三酯。
所述b2)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱和1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸乙醇胺的质量比可为1:0.01～2；
所述b3)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱的质量比可为1:0.01～2；所述c2)中，甘油三酯和胆固醇油酸酯的质量比可为1～5：1。所述b2)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱和1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸乙醇胺的质量比具体可为2:1、1:1或1:2；所述b3)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱的质量比具体可为2:1、1:1或1:2；所述c2)中，甘油三酯和胆固醇油酸酯的质量比具体可为5:1、4:1、3:1或2:1。
所述甘油三酯(triacylglycerol，TAG)的制备方法可如下：(1)取死亡 的SD大鼠1只，取皮下脂肪与大网膜脂肪，剪碎；(2)将步骤(1)得到的碎组织置于离心管中，加入萃脂液甲(氯仿：去离子水＝1：1，v/v)，剧烈涡旋1分钟，然后8000g离心10分钟；(3)取步骤(2)得到的下层有机相，置于新的离心管中，如果发现其浑浊的话按照步骤(2)中的萃取方法进行重复萃取直至其澄清；(4)取步骤(3)得到的下层有机相，在高纯氮下吹干(若吹干过程中发现其变浑浊的话则按照步骤(2)中的萃取方法进行重复萃取)；(5)取步骤(4)得到的下层有机相，在高纯氮下吹干(连续3次称重质量不变)，产物即为甘油三酯。
所述甘油三酯可为甘油三油酸酯(triolein，TO)。所述甘油三油酸酯(triolein,TO)具体可为Sigma公司的产品，产品目录号为92860。
所述磷脂和所述中性脂质的质量比可为(d1)至(d6)中的任一种：(d1)0.25～3:5；(d2)3:5；(d3)2:5；(d4)1:5；(d5)1:10；(d6)1:20。
利用上述脂肪体的制备方法制备的脂肪体也属于本发明的保护范围。
本发明还提供了一种制备人工脂滴的方法。
本发明所提供的制备人工脂滴的方法，包括在通过上述任一所述脂肪体的制备方法制备的脂肪体上招募一种或几种固有蛋白和/或功能蛋白，获得人工脂滴。
所述固有蛋白可为MLDS蛋白、MDT-28蛋白或Perilipin-2蛋白。所述功能蛋白可为ATGL。所述MLDS蛋白可为如下e1)或e2)：e1)氨基酸序列如序列表中序列2所示的蛋白质；e2)将e1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与MLDS蛋白具有相同功能的蛋白质。所述MDT-28蛋白可为如下f1)或f2)：f1)氨基酸序列如序列表中序列4所示的蛋白质；f2)将f1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与MDT-28蛋白具有相同功能的蛋白质。所述Perilipin-2蛋白可为如下g1)或g2)：g1)氨基酸序列如序列表中序列6所示的蛋白质；g2)将g1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与Perilipin-2蛋白具有相同功能的蛋白质。所述ATGL可为如下h1)或h2)：h1)氨基酸序列如序列表中序列8所示的蛋白质；h2)将h1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与ATGL具有相同功能的蛋白质。
利用上述制备人工脂滴的方法制备的人工脂滴也属于本发明的保护范围。
本发明还提供了一种制备人工脂蛋白的方法。
本发明所提供的制备人工脂蛋白的方法，包括在通过上述任一所述脂肪体的制备方法制备的脂肪体上招募一种或几种载脂蛋白，获得人工脂蛋白。
所述载脂蛋白可为Apo A-I。
所述Apo A-I具体可通过参考文献Peitsch,M.C.et al.A purification method for apolipoprotein A-I and A-II.Anal Biochem 178，301-305(1989). 中记载的方法获得。
利用上述制备人工脂蛋白的方法制备的人工脂蛋白也属于本发明的保护范围。
(j1)至(j6)中的任一种也属于本发明的保护范围：(j1)通过上述任一所述脂肪体的制备方法制备的脂肪体在制备人工脂滴和/或人工脂蛋白和/或药物载体中的应用；(j2)通过上述任一所述人工脂滴的制备方法制备的人工脂滴在制备药物载体中的应用；(j3)通过上述任一所述人工脂滴的制备方法制备的人工脂滴在制备药物中的应用；(j4)通过上述任一所述人工脂蛋白的制备方法制备的人工脂蛋白在制备药物载体中的应用；(j5)通过上述任一所述人工脂蛋白的制备方法制备的人工脂蛋白在制备药物中的应用；(j6)通过上述任一所述脂肪体的制备方法制备的脂肪体作为药物载体的应用。
为解决上述技术问题，本发明还提供了一种药物。
本发明所提供的药物，其活性成分为负载有具有药用功能的化合物的脂肪体；所述脂肪体为通过上述任一所述脂肪体的制备方法制备的脂肪体。所述药物可为脂溶性药物。所述脂溶性药物可为能与中性脂质互溶的药物。所述中性脂质具体可为甘油三酯。所述药物具体可为治疗II型糖尿病和/或代谢紊乱的药物或候选药物。上述药物中，所述具有药用功能的化合物可为式a所示的化合物；
Figure PCTCN2016106732-appb-000001
上述药物中，所述具有药用功能的化合物具体可为TOCRIS公司生产的CDN1163，其产品目录号为5869。所述“负载有具有药用功能的化合物的脂肪体”即为实施例3中的载药脂肪体。
所述载药脂肪体的制备方法具体可如下：(1)取10mg TAG加入到微量离心管中，加入100μl氯仿，充分溶解，得到TAG溶液；(2)取2mg CDN 1163，置于另一微量离心管中，加入200μl氯仿，充分溶解，得到浓度为10μg/μl的药物溶液；(3)完成步骤(1)和步骤(2)后，将TAG溶液和药物溶液混合均匀，然后用高纯氮气吹干溶剂，得到混合物质；(4)取80μL DOPC溶液(其中含有2mg DOPC)加入到新的微量离心管中，用高纯氮气吹干溶剂；(5)向完成步骤(4)的微量离心管中加入100μl缓冲液B和6mg步骤(3)得到的混合物质，涡旋4min(涡旋10s，停止10s)，得到乳白色的脂质混合物甲，将该脂质混合物甲以20000g离心5min；离心后，微量离心管中底部为沉淀组分甲，液相体系呈现两层分层(上层为白色带甲，白色带甲以下的部分为溶液甲)；(6)完成步骤(5)后，通过抽取的方式弃除溶液甲与沉淀组分甲，保留白色带甲， 加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物乙，将该脂质混合物乙以20000g离心5min；离心后，如果微量离心管中底部有沉淀，即为沉淀组分乙，液相体系呈现两层分层(上层为白色带乙，白色带乙以下的部分为溶液乙)；(7)完成步骤(6)后，通过抽取的方式弃除溶液乙与沉淀组分乙，保留白色带乙，加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物丙，将该脂质混合物丙以20000g离心5min；离心后，液相体系呈现两层分层(上层为白色带丙，白色带丙以下的部分为溶液丙)。步骤(7)即步骤(6)的重复，实际应用中的步骤(6)的重复次数以白色带下层无沉淀为准。(8)完成步骤(7)后，取白色带丙，加入100μl缓冲液B，涡旋混匀，1000g离心5min，离心后，液相体系呈现两层分层(上层为白色带丁，白色带丁以下的部分为溶液丁)。收集溶液丁，即为载药脂肪体。
本发明提供了一种脂肪体的制备方法及其应用。本发明所提供的一种脂肪体的制备方法通过涡旋混合中性脂质和磷脂，使用两步法纯化，制备出了由中性脂质核心和单层磷脂膜构成的脂肪体。在脂肪体上招募固有蛋白和/或功能蛋白，如细菌的蛋白MLDS，线虫的蛋白MDT-28、哺乳动物的蛋白Perilipin-2、脂肪甘油三酯脂酶(adipose triglyceride lipase,ATGL)，获得人工脂滴；在脂肪体上招募载脂蛋白，如Apo A-I(apolipoprotein A-I)，获得人工脂蛋白。利用本发明提供的方法制备的脂肪体、人工脂滴和人工脂蛋白，可作为药物载体，进而完成各种生物学和医学的目标。
附图说明
图1为使用涡旋及两步法离心制备纯化脂肪体。A为脂肪体制备流程图：a为制备脂肪体的组分；b为制备脂肪体的流程图，蓝色箭头代表涡旋，红色箭头显示被移除的组分。B为脂肪体制备过程中各组分的形态：a为光学显微镜的观察结果(比例尺＝10微米)：箭头表示非球形结构，左图为初始制备组分，中图为沉淀组分，右图为上层组分；b为荧光显微镜的观察结果(比例尺＝10微米)：箭头表示非球形结构，左图为初始制备组分，中图为沉淀组分，右图为上层组分；c为显微镜进行透射电镜正染的结果(比例尺＝1微米)：左图为初始制备组分，中图为沉淀组分，右图为上层组分。C为光学显微镜观察脂肪体的形态(比例尺＝10微米)：左图为微分干涉相衬光学显微镜，右图为荧光显微镜。D为透射电镜正染脂肪体和脂质体的结果(比例尺＝500纳米)：左图为脂肪体，右图为脂质体。
图2为脂肪体的特征。其中A为脂肪体和线粒体的形态(比例尺＝500纳米)：a为透射电镜的观察结果：左图为脂肪体，右图为线粒体；b为冷冻电镜的观察结果：左图为脂肪体，右图为线粒体。B通过动态光散射(DLS)测定脂肪体的尺寸，多分散性指数＝0.085。C为薄层层析色谱分析脂肪体制备的过程中每个组分的DOPC/总脂质的比例：a为碘蒸气对样品染色结果(泳道1为脂质Marker，泳道2为初始制备组分，泳道3为混合组分，泳道4为脂肪体a)；b为各组分 DOPC与总脂质的比例，N＝3，平均值±方差。D为脂肪体与脂滴的形态：a为脂肪体的形态，左图为肉眼观察结果，中图为光学显微镜的观察结果，右图为荧光显微镜的观察结果；b为小鼠肝脏组织-脂滴的形态，左图为肉眼观察结果，中图为光学显微镜的观察结果，右图为荧光显微镜的观察结果；c为小鼠棕色脂肪组织-脂滴的形态，左图为肉眼观察结果，中图为光学显微镜的观察结果，右图为荧光显微镜的观察结果；d为浊红球菌RHA1-脂滴的形态，左图为肉眼观察结果，中图为光学显微镜的观察结果，右图为荧光显微镜的观察结果。
图3制备脂肪体的影响因素。A为不同涡旋时间(1为脂肪体b，2为脂肪体c，3为脂肪体d，4为脂肪体a)对脂肪体的影响：a为脂肪体的形态，b为脂肪体的光密度，c为脂肪体的尺寸；B为不同比例的DOPC和TAG制备的脂肪体的光密度(a)和尺寸(b)；C为不同比例的DOPC和DSPC制备的脂肪体的光密度(a)和尺寸(b)；D为不同比例的DOPC和DOPE制备的脂肪体的光密度(a)和尺寸(b)；E为不同比例的TAG和CO制备的脂肪体的光密度(a)和尺寸(b)；F为4℃或室温下孵育7天脂肪体的光密度(a)和尺寸(b)。
图4为室温或4℃下储存7天的脂肪体的显微镜照片(比例尺＝10微米)。A和C为光学显微镜；B和D为荧光显微镜。
图5为SMT3-Perilipin-2蛋白的分离与纯化。A为用SDS-PAGE分离并通过银染(上图)和Western印迹(下图)分析IPTG诱导前后的全细胞裂解液。黑色箭头指向SMT3-Perilipin-2蛋白对应的条带；B为经镍离子金属亲和层析柱纯化的各组分进行SDS-PAGE和胶体蓝染色的分析结果。C为从镍柱洗脱的SMT3-Perilipin-2蛋白通过分子筛进一步纯化。四个组分(1，2，3和4)根据其保留体积分别收集，图中用蓝色短线分开表示；D为通过SDS-PAGE分析镍柱纯化的SMT3-Perilipin-2蛋白(咪唑洗脱)和分子筛得到的组分。
图6为人工重组的不同物种来源的脂滴固有/结构蛋白被招募到脂肪体上。
图7为通过募集脂滴的固有/结构蛋白构建人工脂滴。A为人工脂滴构建示意图；B为脂肪体可以招募脂滴固有/结构蛋白GFP融合蛋白；C为SMT3-Perilipin-2蛋白饱和度测定。
图8为脂肪体招募脂滴固有/结构蛋白GFP融合蛋白(比例尺＝5微米)。
图9为SMT3-ATGL蛋白在室温或4℃下与脂肪体孵育后的银染分析结果。
图10为脂肪体上募集ATGL与Apo A-I。A为SMT3-ATGL蛋白饱和度测定，其中泳道1至7对应加入的SMT3-ATGL蛋白浓度分别为：0.091，0.132，0.171，0.209，0.244，0.278，0.310微克/微升；B为通过募集高密度脂蛋白的载脂蛋白Apo A-I构建人工脂蛋白。
图11为使用涡旋及两步法离心制备载药脂肪体。A为载药脂肪体制备流程图：a为制备载药脂肪体的组分；b为制备载药脂肪体的流程图；B为制备的载药脂肪体；C通过动态光散射(DLS)测定载药脂肪体的尺寸；D为高效液相色谱检测结果。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明中所述脂肪体(adiposome)定义为一类以天然或人工合成的中性脂质为核心，天然或人工合成的极性脂质单层膜包裹的，人工合成的结构。脂肪体的脂质组成及形态结构与脂滴近似，因此可以作为制备人工脂滴的主要原材料。
1,2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱(DOPC)为Avanti公司的产品，产品状态为DOPC溶液，即DOPC溶解于氯仿中。1,2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸乙醇胺(DOPE)为Avanti公司的产品，产品状态为DOPE溶液，即DOPE溶解于氯仿中。1,2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱(DSPC)为Avanti公司的产品，产品状态为DSPC溶液，即DSPC溶解于氯仿中。胆固醇油酸酯(cholesteryl oleate，CO)为Alfa Aesar公司的产品。尼罗红为Sigma-Aldrich公司的产品。LipidTOX red为Invitrogen公司的产品，产品目录号为H34476。抗Perilipin-2为Abcam公司的产品，产品目录号为ab108323。Percoll为GE Healthcare公司产品。醋酸铀、25％戊二醛和EMbed 812包埋试剂盒均为Electron Microscopy Sciences公司(哈特菲尔德，美国)产品。四氧化锇(电镜级)为NAKALAI Tesque公司(京都，日本)产品。丹宁酸为Sigma公司的产品。C57BL/6小鼠为北京维通利华实验动物技术有限公司产品。动态光散射仪(DLS，Delsa Nano C颗粒分析仪)为Beckman公司产品。
下述实施例中的“涡旋”均使用Vortex-Genie 1涡旋仪(Scientific Industries,Inc.公司，美国)进行，“胶体蓝染色”均使用胶体蓝染色试剂盒(Invitrogen公司的产品，产品目录号为LC6025)进行。
线粒体从C57BL/6小鼠的棕色脂肪组织分离得到，具体分离方法参考Yu，J.et al.Lipid droplet remodeling and interaction with mitochondria in mouse brown adipose tissue during cold treatment.Biochim Biophys Acta 1853，918-928，doi:10.1016/j.bbamcr.2015.01.020(2015).
2.5％戊二醛溶液的制备方法：将25％戊二醛溶解于pH7.4、0.1M PB缓冲液，使戊二醛在体系中质量浓度为2.5％。
实施例1中的甘油三酯(triacylglycerol，TAG)的制备方法如下：(1)取死亡的SD大鼠1只，取皮下脂肪与大网膜脂肪，剪碎；(2)将步骤(1)得到的碎组织置于离心管中，加入萃脂液甲(氯仿：去离子水＝1：1，v/v)，剧烈涡旋1分钟，然后8000g离心10分钟；(3)取步骤(2)得到的下层有机相，置于新的离心管中，如果发现其浑浊的话按照步骤(2)中的萃取方法进行重复萃取直至其澄清；(4)取步骤(3)得到的下层有机相，在高纯氮下吹干，(若 吹干过程中发现其变浑浊的话则按照步骤(2)中的萃取方法进行重复萃取)；(5)取步骤(4)得到的下层有机相，在高纯氮下吹干(连续3次称重质量不变)，产物即为甘油三酯。
缓冲液B的溶质及其在缓冲液中浓度为：20mM HEPES(实际应用中15mM-25mM HEPES均可)，100mM KCl(实际应用中80mM-120mM KCl均可)，2mM MgCl2(实际应用中1.5-2.5mM MgCl2均可)；溶剂为去离子水；pH为7.4(实际应用中7.2-7.6均可)。
脂质体为磷脂酰胆碱和磷脂酰乙醇胺按质量比3:7混合，然后用带有100nm孔径的聚碳酸酯滤器微型挤出机(Avanti公司产品)制备的单层脂质体，具体制备方法参考Nakatogawa，H.，Ichimura，Y.& Ohsumi，Y.Atg8，a ubiquitin-like protein required for autophagosome formation，mediates membrane tethering and hemifusion.Cell 130，165-178，doi:10.1016/j.cell.2007.05.021(2007).
用荧光显微镜对脂肪体或脂滴进行观察，具体步骤如下：将脂肪体或脂滴用尼罗红(浓度为1μg/ml)或LipidTOX red稀释液(LipidTOX red用缓冲液B以1：1000倍稀释，得到LipidTOX red稀释液)染色，室温孵育30min，取孵育后的脂肪体或脂滴6μl，滴加在载玻片上，然后加2μl抗荧光淬灭剂(碧云天公司产品，产品目录号为P0126)混合、盖盖玻片。用Zeiss M2荧光显微镜、奥林巴斯FV1000荧光共聚焦显微镜或DeltaVision OMX V3超分辨率显微镜获得荧光图像。
透视电镜正染具体步骤如下：取脂肪体或脂质体8μl滴在辉光放电处理后的覆有碳膜的铜网上，静置1min，用滤纸吸干多余的样品，然后将样品用1％四氧化锇固定10min、去离子水漂洗，再将样品分别用0.1％单宁酸和2％乙酸双氧铀依次染色5min、用去离子水漂洗。用Tecnai Spirit(FEI)透射型电子显微镜在100千伏下获得显微照片。
用透视电镜对脂肪体或线粒体进行观察，具体步骤如下：取脂肪体或线粒体迅速与融化的3％低熔点琼脂糖混合，并在冰上凝固，然后切成约1mm3的小块。将小块在2.5％戊二醛溶液中固定30min，然后继续用溶于pH7.4、0.1M PB缓冲液的1％四氧化锇在室温下固定1h，得到固定块。将该固定块用去离子水洗涤，用乙醇脱水，然后用EMbed 812包埋试剂盒进行渗透和包埋，并在60℃下聚合24h，用Leica EM UC6超微切片机制备70nm超薄切片。用Tecnai Spirit(FEI)透射型电子显微镜在100千伏下获得显微照片。
用冷冻电镜对脂肪体或线粒体进行观察，具体步骤如下：取脂肪体或线粒体4μl，滴加在铜网上。使用Vitrobot Mark IV(FEI公司产品)在100％湿度下吸干3秒，然后快速浸入预先用液氮冷却的液态乙烷中使其玻璃化。使用装有Gatan UltraScan4000相机(产品型号895)的Titan Krios冷冻电子显微镜(FEI公司产品)在300千伏下拍摄显微照片。
缓冲液T为pH7.4、含50mM Tris-HCl和150mM NaCl的缓冲液。
载体pET28a为Novagen公司产品。载体pGEX-6p-1购自Novagen公司。镍离子金属亲和层析柱填料为螯合有镍离子的Chelating Sepharose Fast Flow，为Amersham Biosciences公司产品。GST亲和层析柱填料为生工生物工程(上海)有限公司产品。
“改造后的pET28a质粒”记载于如下文献中：Hu,W.,Wu,H.,Zhang,H.,Gong,W.& Perrett,S.Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.Biomol NMR Assign 9,329-332,doi:10.1007/s12104-015-9603-5(2015)。“改造后的pET28a质粒”在下文中命名为载体pET28a-SMT3。
实施例1、脂肪体的制备
一、制备脂肪体a
1、利用运用涡旋和两步法离心制备脂肪体a，具体步骤如下：
(1)取80μL DOPC溶液(其中含有2mg DOPC)加入到微量离心管中，用高纯氮气吹干溶剂。
(2)完成步骤(1)后，向微量离心管中加入100μl缓冲液B和5mg TAG，涡旋4min(涡旋10s，停止10s)，得到乳白色的脂质混合物1(即初始制备组分)，将该脂质混合物1以 20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，微量离心管中底部为沉淀组分1，液相体系呈现两层分层(上层为白色带1，白色带1以下的部分为溶液1)。
(3)完成步骤(2)后，通过抽取的方式弃除溶液1与沉淀组分1，保留白色带1，加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物2，将该脂质混合物2以20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，如果微量离心管中底部有沉淀，即为沉淀组分2，液相体系呈现两层分层(上层为白色带2，白色带2以下的部分为溶液2)。
(4)完成步骤(3)后，通过抽取的方式弃除溶液2与沉淀组分2，保留白色带2，加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物3，将该脂质混合物3以20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，液相体系呈现两层分层(上层为白色带3，白色带3以下的部分为溶液3)。
步骤(4)即步骤(3)的重复，实际应用中的步骤(3)的重复次数以白色带下层无沉淀为准。
(5)完成步骤(4)后，取白色带3，加入100μl缓冲液B，涡旋混匀，1000g离心5min(实际应用中800-1200g离心3-7min)，离心后，液相体系呈现两层分层(上层为白色带4，白色带4以下的部分为溶液4)。收集溶液4，即为脂肪体a。
将上述步骤中的白色带4命名为上层组分。将上述步骤中的沉淀组分1和沉淀组分2混合，命名为沉淀组分。将上述步骤中的沉淀组分和上层组分混合，命名为混合组分。
利用上述方法制备脂肪体a的具体流程见图1中A(其中a为制备脂肪体a 的组分，b为制备脂肪体a的流程图)。对制备脂肪体a过程中的组分形态用光学显微镜和荧光显微镜进行观察、用显微镜进行透射电镜正染，结果如下：初始制备组分、沉淀组分和上层组分中除含中性脂质的球状结构，还存在许多其它类型的结构(图1中B)；溶液4几乎所有结构均为球形，且均为尼罗红阳性，而且尼罗红标记的结构的尺寸与光学显微镜图像中的球状结构的尺寸一致，这表明这些球状结构都含有TAG核心(图1中C)；将脂肪体a和脂质体用显微镜进行透射电镜正染，结果表明两者具有明显的差异(图1中D)。
2、脂肪体a的特征
(1)用透射电镜分别对脂肪体a和线粒体进行观察，用冷冻电镜分别对脂肪体a和线粒体进行观察，结果表明(图2中A)，脂肪体a由单层磷脂膜包裹，线粒体由双层磷脂膜包裹。
(2)用动态光散射仪测定脂肪体a的平均尺寸约为189纳米(图2中B)，然后使用文献(Ding Y，Zhang S，et al.Isolating lipid droplets from multiple species.Nat Protoc.2013 Jan；8(1):43-51.doi:10.1038/nprot.2012.142.Epub 2012 Dec 6.)中的公式，计算得到脂肪体a中DOPC与总脂质(总脂质即TAG和DOPC)的体积比约为6.6％。
(3)使用薄层色谱法分析脂质，具体步骤如下：
a、取待提取物(待提取物体积和下述萃脂液乙中缓冲液B的体积之和为300μl)，用萃脂液乙(由300μl氯仿、300μl甲醇和缓冲液B组成)进行萃取以获取脂质(收集有机相，命名为有机相甲)，剩余的部分再加入300μl氯仿进行萃取以进一步获取脂质(收集有机相，命名为有机相乙)；将有机相甲和有机相乙混合，用氮气吹干，得到待提取物的总脂质；待提取物为脂肪体a、脂质混合物1(即初始制备组分)或混合组分。
b、将步骤a得到的总脂质溶解在100μl氯仿中，上样10μl至硅胶板；
c、完成步骤b后，样品在正己烷：乙醚：冰乙酸(体积比为80:20:1)的展开剂中展开，以分离TAG；
d、完成步骤c后，在空气中挥干有机溶剂(即步骤c中的正己烷、乙醚和冰乙酸)，将硅胶板再次在氯仿：甲醇：冰乙酸：水(体积比为75:13:9:3)的溶剂系统中展开，以分离DOPC；
e、完成步骤d后，用饱和碘蒸气进行染色，用ImageJ软件对脂质斑点的灰度进行量化。
结果表明(图2中C)，样品中DOPC与脂质的比例为7.1±1.2％，与步骤(2)中的计算值相似，表明脂肪体a具有单层磷脂膜的结构。
(4)根据Ding Y，Zhang S，et al.Isolating lipid droplets from multiple species.Nat Protoc.2013Jan；8(1)：43-51.doi：10.1038/nprot.2012.142.Epub 2012Dec 6.)和Yu，J.et al.Lipid droplet remodeling and interaction with mitochondria in mouse brown adipose tissue during cold  treatment.Biochim Biophys Acta 1853,918-928，doi：10.1016/j.bbamcr.2015.01.020(2015)中记载的方法分别从C57BL/6小鼠肝脏组织、C57BL/6小鼠棕色脂肪组织和产油菌RHA1中分离脂滴，依次命名为肝脏组织-脂滴(Liver LD)、棕色脂肪组织-脂滴(BAT LD)和RHA1-脂滴(RHA1 LD)。
脂肪体a、RHA1 LD、Liver LD和BAT LD均呈牛奶状溶液。用动态光散射仪测量脂肪体a、RHA1 LD、Liver LD和BAT LD的平均尺寸，结果BAT LD的粒径最大，平均直径为1848nm；RHA1 LD的粒径最小，平均直径为493nm；脂肪体a的平均直径为189nm(图2中B)。上述结果与光学显微镜得到的结果基本一致(图2中D)。用荧光显微镜对脂肪体或脂滴进行观察，脂肪体a、RHA1 LD、Liver LD和BAT LD都呈现球状结构(图2中D)，表明它们都含有TAG核心。
3、影响脂肪体形成的因素
为优化制备脂肪体的条件，我们系统地改变了步骤1中所述涡旋和两步法中两个主要因素，即步骤(2)中的涡旋时间和两种原料(磷脂与中性脂质)的比例，并通过测量在600nm波长处的光密度(OD600)来反映脂肪体的产量，通过动态光散射仪测量脂肪体的尺寸。
(1)涡旋时间的优化
按照上述步骤1的方法，将步骤1中(1)的涡旋4min分别替换为涡旋1min、涡旋2min和涡旋3min，其它步骤均不变，得到脂肪体b、脂肪体c和脂肪体d，然后分别测量脂肪体a、脂肪体b、脂肪体c或脂肪体d的尺寸和光密度。
实验结果见图3中A。结果表明，脂肪体的产量随涡旋时间的延长而上升，而脂肪体尺寸随涡旋时间的延长而降低，在大约2min达到最小值并进入平台。综合脂肪体的光密度较高(即产量较高)且脂肪体的尺寸较大两个因素，确定涡旋和两步法中步骤(2)的最佳涡旋时间为4min。
(2)磷脂与中性脂质的比例优化
制备脂肪体a中的磷脂具体为DOPC，中性脂质具体为TAG，DOPC:TAG的比例为2:5。
①按照上述步骤1的方法，将步骤1中(1)的2mg DOPC分别替换为0.25mg DOPC、0.5mg DOPC、1mg DOPC和3mg DOPC，其它步骤均不变，得到脂肪体a1(DOPC:TAG的比例为1:20)、脂肪体a2(DOPC:TAG的比例为1:10)、脂肪体a3(DOPC:TAG的比例为1:5)和脂肪体a4(DOPC:TAG的比例为3:5)，然后分别测量脂肪体a1、脂肪体a2、脂肪体a3和脂肪体a4的尺寸和光密度。
实验结果见图3中B。结果表明，当DOPC与TAG的比例为2：5时，脂肪体的产量最高；脂肪体的尺寸随着DOPC与TAG的比例上升而减小，至DOPC:TAG为1：5时达到最小并进入平台。综合脂肪体的即产量较高且脂肪体的尺寸较大两个因素，确定制备脂肪体中DOPC与TAG的最佳比例为2:5。
②按照上述步骤1的方法，将步骤1中(1)的2mg DOPC分别替换为1.33mg DOPC和0.67mgDSPC、1mg DOPC和1mgDSPC、0.67mg DOPC和1.33mg DSPC、以及2mg DSPC，其它步骤均不变，得到脂肪体A1(DOPC:DSPC的比例为2:1)、脂 肪体A2(DOPC:DSPC的比例为1:1)、脂肪体A3(DOPC:DSPC的比例为1:2)和脂肪体A4(磷脂仅为DSPC)，然后分别测量脂肪体A1、脂肪体A2、脂肪体A3和脂肪体A4的尺寸和光密度。
结果表明，当DOPC与DSPC的比例降低时，即DSPC的相对含量升高时，脂肪体的产量明显下降(图3中C的a)，而脂肪体的尺寸随着DOPC与DSPC的比例降低而急剧增大(图3中C的b)。
③按照上述步骤1的方法，将步骤1中(1)的2mg DOPC分别替换为1.33mg DOPC和0.67mgDOPE、1mg DOPC和1mgDOPE、0.67mg DOPC和1.33mgDOPE、和、2mgDOPE，其它步骤均不变，得到脂肪体B1(DOPC:DOPE的比例为2:1)、脂肪体B2(DOPC:DOPE的比例为1:1)、脂肪体B3(DOPC:DOPE的比例为1:2)和脂肪体B4(磷脂仅为DOPE)，然后分别测量脂肪体B1、脂肪体B2、脂肪体B3和脂肪体B4的尺寸和光密度。
结果表明，DOPE的掺入不影响脂肪体的产量，直至DOPC与DOPE的比例达到1:2时，脂肪体的产量才急剧下降(图3中D的a)，而脂肪体的尺寸随着DOPC与DOPE的比例降低而急剧增大，直至DOPC与DOPE的比例达到1:2时，脂肪体的尺寸无法检出(图3中D的b)。
④按照上述步骤1的方法，将步骤1中(1)的5mg TAG分别替换为4.17mg TAG和0.83mgCO、4mg TAG和1mgCO、3.75mg TAG和1.25mgCO、和、3.33mg TAG和1.67mgCO，其它步骤均不变，得到脂肪体C1(TAG:CO的比例为5:1)、脂肪体C2(TAG:CO的比例为4:1)、脂肪体C3(TAG:CO的比例为3:1)和脂肪体C4(TAG:CO的比例为2:1)，然后分别测量脂肪体C1、脂肪体C2、脂肪体C3和脂肪体C4的尺寸和光密度。
结果表明，CO的掺入不影响脂肪体的产量，直至TAG与CO的比例达到3:1时，脂肪体的产量才显著下降(图3中E的a)，脂肪体的尺寸也略有下降(图3中E的b)，表明中性脂质组成可以极大地影响脂肪体产量和大小。
4、脂肪体的稳定性
在室温或4℃条件下孵育脂肪体a，孵育7天。整个孵育过程中，每天测定脂肪体a的尺寸、在OD600处的光密度，并用光学显微镜和荧光显微镜对脂肪体进行观察。
结果表明(图3中F和图4)，整个孵育过程中，脂肪体a的光密度和尺寸均无显著变化，可见脂肪体a是相对稳定的。
实施例2、通过招募固有蛋白和/或功能蛋白的重组人工脂滴和通过招募载脂蛋白重组人工脂蛋白
下述实施例中的Ulp1记载在如下文献中：Hu，W.,Wu,H.,Zhang,H.,Gong,W.& Perrett，S.Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1from Saccharomyces cerevisiae.Biomol NMR Assign9，329-332，doi:10.1007/s12104-015-9603-5(2015)。在下述实验孵育过程中Ulp1可剪切蛋白上的SMT3标签。
脂滴是高度动态的，并通过多种脂滴蛋白的介导参与数目繁多的生理功能和相互作用。脂滴上的蛋白可大致分为两类：固有蛋白和动态蛋白。固有蛋白被认为选择性的定位于脂滴上，参与脂滴大小的控制，并介导动态蛋白的定位。例如，磷酸化的PLIN1促使激素敏感性脂肪酶(hormone-sensitive lipase，HSL)定位于脂滴上。因此，使用脂肪体来解构脂滴的功能，就必须在脂肪体上装载脂滴固有蛋白，然后在成功招募固有蛋白生成人工脂滴的基础上，进一步招募功能蛋白。对于脂蛋白而言，不同的载脂蛋白不仅是不同脂蛋白的特异性标记，同时也是脂蛋白的骨架之一。本发明中，脂滴固有蛋白具体为来自于从细菌到哺乳动物的不同生物体的脂滴固有蛋白，包括从产油菌RHA1中分离的RHA1-脂滴(RHA1LD)上的MLDS蛋白，线虫脂滴上的MDT-28蛋白和哺乳动物非脂肪细胞中的Perilipin-2蛋白，功能蛋白为ATGL。载脂蛋白具体为来自于人的载脂蛋白A1(apolipoprotein A-I，Apo A-I)。
一、蛋白的表达和纯化
人工合成序列表中序列1所示的DNA分子(即MLDS基因)，序列1所示的DNA分子编码序列2所示的蛋白质(即MLDS蛋白，NCBI编号为WP_005261062.1)。人工合成序列表中序列3所示的DNA分子(即MDT-28基因，NCBI编号为NM_001129054.2)，序列3所示的DNA分子编码序列4所示的蛋白质(即MDT-28蛋白，NCBI编号为NP_001122526.1)。人工合成序列表中序列5所示的DNA分子(即Perilipin-2基因)，序列5所示的DNA分子编码序列6所示的蛋白质(即Perilipin-2蛋白，NCBI编号为NP_001113.2)。人工合成序列表中序列7所示的DNA分子(即ATGL基因)，序列7所示的DNA分子编码序列8所示的蛋白质(即ATGL，NCBI编号为NP_065109.1)。人工合成序列表中序列9所示的DNA分子(即MLDS-GFP融合基因)，序列9所示的DNA分子编码序列10所示的蛋白质(即MLDS-GFP融合蛋白)。人工合成序列表中序列11所示的DNA分子(即MDT-28-GFP融合基因)，序列11所示的DNA分子编码序列12所示的蛋白质(即MDT-28-GFP融合蛋白)。人工合成序列表中序列13所示的DNA分子(即Perilipin-2-GFP融合基因)，序列13所示的DNA分子编码序列14所示的蛋白质(即Perilipin-2-GFP融合蛋白)。牛血清白蛋白购自Sigma公司，产品目录号为A4612。
1、SMT3-Perilipin-2蛋白的表达与纯化
(1)用序列表中序列5所示的DNA分子替换载体pET28a-SMT3的限制性内切酶EcoRI和XhoI识别序列间的片段(载体pET28a-SMT3被限制性核酸内切酶EcoRI和XhoI切成一个大片段和一个小片段，将大片段与序列5中所示的DNA分子连接)，得到重组质粒。该重组质粒可表达N-末端融合有6×His标签和SMT3结构域的可溶性蛋白。
(2)将步骤(1)构建的重组质粒导入大肠杆菌BL21(DE3)，得到含有重组质粒的重组大肠杆菌。
(3)将步骤(2)获得的重组大肠杆菌接种于2×YT培养基(将16g蛋白胨、 10g酵母提取物和4g氯化钠溶解于1000mL蒸馏水)，37℃培养，当重组大肠杆菌的培养菌液的OD600达到0.6(将该培养菌液命名为诱导前菌液)，加入异丙基β-D-1-硫代半乳糖苷(IPTG)，使IPTG在体系中浓度为0.4mM，16℃诱导24h(IPTG诱导后的培养菌液命名为诱导后菌液)。
分别收集(4000rpm、离心20min)诱导前菌液或诱导后菌液的菌体，重悬于缓冲液T中，然后用高压细胞破碎仪(型号为JNBIO JN-3000 PLUS)裂解，得到全细胞裂解液。将全细胞裂解液进行SDS-PAGE，然后通过银染和Western印迹法进行分析。结果表明(图5中A，其中a为银染分析结果，b为Western印迹法分析结果，箭头指向SMT3-Perilipin-2蛋白对应的条带)，诱导后菌液中SMT3-Perilipin-2蛋白大量表达。
(4)取步骤(3)的诱导后菌液的菌体的全细胞裂解液，30000g离心50min，得到的上清和沉淀，将上清上样至镍离子金属亲和层析柱(填料为螯合有镍离子的Chelating Sepharose Fast Flow，柱体购自Thermo，产品目录号为29924，柱体积4mL，内径1.2cm)，然后先用含20mM咪唑的缓冲液T洗脱12个柱体积以去除非目的蛋白一次，再用含500mM咪唑的缓冲液T洗脱3个柱体积以收集目的蛋白(过柱后全部缓冲液T均含目的蛋白)两次，然后用离心超滤管(Amicon公司产品)进行缓冲液交换去除咪唑，通过尺寸排阻色谱法(Size Exclusive Chromatography,SEC)再次纯化(HiLoad 16/600Superdex 200柱，GE公司产品)，洗脱流速为0.8mL/min，根据其保留体积分别收集四个组分，分别命名为组分1(保留体积为45mL-57mL)、组分2(保留体积为57mL-67mL)、组分3(保留体积为67mL-79mL)和组分4(保留体积为79mL-94mL)。
将镍离子金属亲和层析柱纯化的各组分的进行SDS-PAGE，然后用胶体蓝染色，结果见图5中B(泳道依次为全细胞裂解液、上清、沉淀、流穿、咪唑洗脱液-1(20mM咪唑洗脱液)、咪唑洗脱液-2(500mM咪唑洗脱液第一部分)、咪唑洗脱液-3(500mM咪唑洗脱液第二部分)。尺寸排阻色谱法分子筛纯化结果见图5中C(峰1为组分1,峰2为组分2，峰3为组分3，峰4为组分4)。对全部500mM咪唑二次洗脱液(即咪唑洗脱液-2合并咪唑洗脱液-3)(图5中D的左图)和尺寸排阻色谱法纯化得到的四个组分(组分1、组分2、组分3和组分4)(图5中D的右图)进行SDS-PAGE和胶体蓝染色，结果表明，组分1包含了纯化后的SMT3-Perilipin-2蛋白。
2、MLDS蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为序列表中序列1所示的DNA分子、载体pET28a-SMT3替换为载体pGEX-6p-1、限制性内切酶XhoI替换为BamHI，步骤1中(4)的镍离子金属亲和层析柱替换为GST亲和层析柱、咪唑替换为还原性谷胱甘肽，其它步骤均不变，获得纯化MLDS蛋白。
3、MDT-28蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为 序列表中序列3所示的DNA分子、载体pET28a-SMT3替换为载体pGEX-6p-1、限制性内切酶EcoRI和XhoI替换为限制性内切酶BamHI和NotI，步骤1中(4)的镍离子金属亲和层析柱替换为GST亲和层析柱、咪唑替换为还原性谷胱甘肽，其它步骤均不变，获得纯化MDT-28蛋白。
4、MLDS-GFP融合蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为序列表中序列9所示的DNA分子、载体pET28a-SMT3替换为载体pET28a，其它步骤均不变，获得纯化MLDS-GFP融合蛋白。
5、MDT-28-GFP融合蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为序列表中序列11所示的DNA分子、载体pET28a-SMT3替换为载体pET28a、限制性内切酶EcoRI替换为限制性内切酶BamHI，其它步骤均不变，获得纯化MDT-28-GFP融合蛋白。
6、Perilipin-2-GFP融合蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为序列表中序列13所示的DNA分子、载体pET28a-SMT3替换为载体pET28a，其它步骤均不变，获得纯化Perilipin-2-GFP融合蛋白。
7、SMT3-ATGL蛋白的表达与纯化
按照上述方法，将步骤1中(1)的序列表中序列5所示的DNA分子替换为序列表中序列7所示的DNA分子，步骤1中(2)的大肠杆菌BL21(DE3)替换为大肠杆菌Rosetta，其它步骤均不变，获得纯化SMT3-ATGL蛋白。
8、Apo A-I的表达与纯化
参考文献Peitsch,M.C.et al.A purification method for apolipoprotein A-I and A-II.Anal Biochem 178,301-305(1989).中记载的方法，获得纯化Apo A-I。
二、脂肪体对固有蛋白和/或功能蛋白和/或载脂蛋白的招募
1、固有蛋白的招募与分布
(1)Perilipin-2蛋白的招募
a1、5μg SMT3-Perilipin-2蛋白、25ng Ulp1和实施例1制备的50μl脂肪体a混合，得到100μl的混合体系。
a2、完成步骤a1后，室温下(实际操作中在冰上也可)孵育1h,得到混合体系。
a3、完成步骤a2后，取混合体系，20000g、离心5min，液相体系呈现两层分层(上层为脂肪体)，抽去下层溶液，保留上层。
a4、取完成步骤a3的上层，重悬于100μl缓冲液B中，20000g、离心5min，液相体系呈现两层分层(上层为脂肪体)，抽去下层溶液，保留上层。
a5、取完成步骤a4的上层，重悬于100μl缓冲液B中，20000g、离心5min，液相体系呈现两层分层，抽去下层溶液，保留上层(即脂肪体)。
将5μg SMT3-Perilipin-2蛋白、步骤a5得到脂肪体和步骤a3抽去的下层溶液均进行SDS-PAGE后银染检测。
结果见图6中的泳道7、8和9(泳道7为总蛋白(SMT3-Perilipin-2蛋白)，泳道8为脂质体募集蛋白(步骤a5得到脂肪体)，泳道9为溶液蛋白(步骤a3抽去的下层溶液))。结果表明，Perilipin-2蛋白有约50％被招募到脂肪体a上。将招募有Perilipin-2蛋白的脂肪体命名为人工脂滴Perilipin-2。
蛋白饱和度测定：将SMT3-Perilipin-2蛋白、25ng Ulp1和实施例1中制备的30μl脂肪体a混合，得到50μl的混合体系，混合体系中SMT3-Perilipin-2蛋白的浓度为0.015μg/μl、0.030μg/μl、0.056μg/μl、0.080μg/μl、0.101μg/μl、0.137μg/μl、0.166μg/μl或0.191μg/μl。然后按照上述步骤a2至a5，获得人工脂滴，进行SDS-PAGE，然后用胶体蓝染色，或以Perilipin-2抗体为一抗进行Western blot检测。结果见图7中C(其中a为胶体蓝染色结果，b为Western blot检测结果)。结果表明，Perilipin-2蛋白在脂肪体上的募集是可以饱和的。
(2)MLDS蛋白的招募
b1、5μg MLDS蛋白和实施例1制备的50μl脂肪体a混合，得到100μl的混合体系。
b2、完成步骤b1后，室温下(实际操作中在冰上也可)孵育1h,得到混合体系。
b3、完成步骤b2后，取混合体系，20000g、离心5min，液相体系呈现两层分层(上层为脂肪体)，抽去下层溶液，保留上层。
b4、取完成步骤b3的上层，重悬于100μl缓冲液B中，20000g、离心5min，液相体系呈现两层分层(上层为脂肪体)，抽去下层溶液，保留上层。
b5、取完成步骤b4的上层，重悬于100μl缓冲液B中，20000g、离心5min，液相体系呈现两层分层，抽去下层溶液，保留上层(即脂肪体)。
将5μg MLDS蛋白、步骤b5得到脂肪体和步骤b3抽去的下层溶液均进行SDS-PAGE后银染检测。
结果见图6中的泳道1、2和3(泳道1为总蛋白(MLDS蛋白)，泳道2为脂质体募集蛋白(步骤b5得到脂肪体)，泳道3为溶液蛋白(步骤b3抽去的下层溶液))。结果表明，MLDS蛋白有约80％被招募到脂肪体a上。将招募有MLDS蛋白的脂肪体命名为人工脂滴MLDS。
(3)MDT-28蛋白的招募
将步骤(2)中的MLDS蛋白替换为MDT-28蛋白，其他步骤均不变。结果见图6中的泳道4、5和6(泳道4为总蛋白(MDT-28蛋白)，泳道5为脂质体募集蛋白，泳道6为溶液蛋白)。结果表明，MDT-28蛋白有约90％被招募到脂肪体a上。将招募有MDT-28蛋白的脂肪体命名为人工脂滴MDT-28。
(4)牛血清白蛋白的招募
将步骤(2)中的MLDS蛋白替换为牛血清白蛋白，其他步骤均不变。结果见图6中的泳道11、12和13(泳道11为总蛋白(牛血清白蛋白)，泳道12为脂质体募集蛋白，泳道13为溶液蛋白)。结果表明，牛血清白蛋白没有被招募到脂肪体a上。
将招募有蛋白质的脂肪体命名为人工脂滴。
(5)固有蛋白在人工脂滴上的分布
将步骤 1中(1)5μg SMT3-Perilipin-2蛋白替换为 10μg纯化Perilipin-2-GFP融合蛋白，其他步骤均不变，获得人工脂滴Perilipin-2-GFP。将步骤1中(2)5μg MLDS蛋白替换为10μg纯化MLDS-GFP融合蛋白，获得人工脂滴MLDS-GFP。将步骤1中(3)5μg MDT-28蛋白替换为10μg MDT-28-GFP融合蛋白，获得人工脂滴MDT-28-GFP。
然后用共聚焦显微镜对人工脂滴进行观察。结果表明(图7中B和图8)，人工脂滴上的荧光蛋白质呈环形结构，Perilipin-2-GFP融合蛋白、MLDS-GFP融合蛋白和MDT-28-GFP融合蛋白均匀分布在人工脂滴的表面上，偶尔可观察到大的荧光蛋白的聚集体，未检测到游离的GFP；使用OMX DeltaVision(SIM)超分辨率显微镜观察人工脂滴，人工脂滴上的蛋白仍然呈现均匀分布的状态。
2、功能蛋白ATGL的招募
将步骤1中(1)SMT3-Perilipin-2蛋白替换为SMT3-ATGL蛋白，50μl脂肪体a替换为30μl脂肪体a，其他步骤均不变，获得招募有ATGL的脂肪体。
结果见图9(左图为常温孵育的实验结果，右图为4℃孵育的实验结果，其中泳道1和5为Marker，泳道2和6为总蛋白(SMT3-ATGL蛋白)，泳道3和7为脂质体募集蛋白，泳道4和8为溶液蛋白)。结果表明，ATGL有约1/3被招募到脂肪体a上。将招募有ATGL的脂肪体命名为人工脂滴ATGL。
蛋白饱和度测定：将SMT3-ATGL蛋白和实施例1中制备的50μl脂肪体a混合，得到100μl的混合体系，混合体系中SMT3-ATGL蛋白的浓度为0.091μg/μl，0.132μg/μl，0.171μg/μl，0.209μg/μl，0.244μg/μl，0.278μg/μl或0.310μg/μl。然后按照步骤1中的a2至a5，获得人工脂滴，进行SDS-PAGE，然后银染，或以ATGL抗体(Cell Signaling Technology公司，产品目录号为2138)为一抗进行Western blot检测。结果见图10中A(其中a为银染结果，b为Western blot检测结果)。结果表明，当ATGL在反应体系中的浓度为0.209μg/μl时，脂肪体a对ATGL的招募达到了饱和。
3、载脂蛋白的招募
将步骤1中(2)5μg MLDS蛋白替换为9μg Apo A-I，其他步骤均不变，获得招募有Apo A-I的脂肪体。结果见图10中B(泳道1为脂质体募集蛋白，泳道2为溶液蛋白，泳道3为总蛋白(Apo A-I))。结果表明，Apo A-I有约50％被招募到脂肪体a上。将招募有Apo A-I的脂肪体命名为人工脂蛋白Apo A-I。
制备脂肪体a’，方法基本同实施例1的步骤一，差别仅在于用商购的甘油三油酸酯代替实施例1中的甘油三脂，在步骤(5)中得到脂肪体a’。将脂肪体a’进行实施例2的各项试验，其结果与脂肪体a基本一致，均无显著差异。
商购的甘油三油酸酯为甘油三油酸酯(triolein,TO)，购自Sigma公司，产品目录号为92860。
实施例3、脂肪体在作为药物载体中的应用
本实施例中的药物为式a所示的化合物；
Figure PCTCN2016106732-appb-000002
该药物为脂溶性药物，能与甘油三酯互溶，为用于治疗II型糖尿病和/或代谢紊乱的候选药物，具体为TOCRIS公司的产品目录号为5869的产品，商品名称为CDN 1163。ZORBAX SB-C18色谱柱为Agilent公司的产品。
一、制备载药脂肪体
1、利用运用涡旋和两步法离心制备载药脂肪体，具体步骤如下：
(1)取10mg TAG加入到微量离心管中，加入100μl氯仿，充分溶解，得到TAG溶液。
(2)取2mg CDN 1163，置于另一微量离心管中，加入200μl氯仿，充分溶解，得到浓度为10μg/μl的药物溶液。
(3)完成步骤(1)和步骤(2)后，将TAG溶液和药物溶液混合均匀，然后用高纯氮气吹干溶剂，得到混合物质(混合物质中含10mg TAG和2mg CDN1163)。
(4)取80μL DOPC溶液(其中含有2mg DOPC)加入到微量离心管中，用高纯氮气吹干溶剂。
(5)向完成步骤(4)的微量离心管中加入100μl缓冲液B和6mg步骤(3)得到的混合物质，涡旋4min(涡旋10s，停止10s)，得到乳白色的脂质混合物甲，将该脂质混合物甲以20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，微量离心管中底部为沉淀组分甲，液相体系呈现两层分层(上层为白色带甲，白色带甲以下的部分为溶液甲)。
(6)完成步骤(5)后，通过抽取的方式弃除溶液甲与沉淀组分甲，保留白色带甲，加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物乙，将该脂质混合物乙以20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，如果微量离心管中底部有沉淀，即为沉淀组分乙，液相体系呈现两层分层(上层为白色带乙，白色带乙以下的部分为溶液乙)。
(7)完成步骤(6)后，通过抽取的方式弃除溶液乙与沉淀组分乙，保留白色带乙，加入100μl缓冲液B，涡旋，得到乳白色的脂质混合物丙，将该脂质混合物丙以20000g离心5min(实际应用中18000-22000g离心3-7min均可)。离心后，液相体系呈现两层分层(上层为白色带丙，白色带丙以下的部分为溶液丙)。
步骤(7)即步骤(6)的重复，实际应用中的步骤(6)的重复次数以白色 带下层无沉淀为准。
(8)完成步骤(7)后，取白色带丙，加入100μl缓冲液B，涡旋混匀，1000g离心5min(实际应用中800-1200g离心3-7min)，离心后，液相体系呈现两层分层(上层为白色带丁，白色带丁以下的部分为溶液丁)。收集溶液丁，即为载药脂肪体。
利用上述方法制备载药脂肪体的具体流程见图11中A(其中a为制备载药脂肪体的组分，b为制备载药脂肪体的流程图)。利用上述方法制备的载药脂肪体见图11中B(左为实施例1步骤一中1制备的脂肪体a，右为载药脂肪体)。
二、载药脂肪体的特征
1、用动态光散射仪测定脂肪体a的平均尺寸约为166.1纳米(图11中C)。
2、测定载药脂肪体中CDN1163的浓度。具体步骤如下：
(1)标准曲线的绘制
准确称取CDN 1163 10mg于100ml容量瓶中，用无水甲醇(色谱级)溶解并定容至100ml，配成100μg/ml CDN 1163母液。然后继续用无水甲醇(色谱级)稀释获得浓度分别为20、4μg/mL的标准溶液。使用配有ZORBAX SB-C18色谱柱(5.0μm，4.6mm×150mm)的Agilent 1260液相色谱仪测定不同浓度CDN 1163的峰面积，3次重复。流动相由甲醇和水组成，甲醇和水的体积比为85：15，流速为1.0mL/min。检测波长为245nm。
以CDN1163的浓度为横坐标，峰面积为纵坐标，绘制标准曲线。标准曲线的线性方程为：y＝61.796x+18.2(R2＝0.99999)，其中y为峰面积，x为CDN 1163浓度(μg/mL)。CDN 1163浓度在0-100μg/ml线性关系良好。
(2)测定载药脂肪体中CDN 1163的浓度
取载药脂肪体10μl，加入990μl无水甲醇(色谱级)，涡旋10s(目的为充分混合)，然后20000g离心5min，弃沉淀，收集上清液。使用配有ZORBAX SB-C18色谱柱(5.0μm，4.6mm×150mm)的Agilent 1260液相色谱仪测定上清液的峰面积，3次重复。流动相由甲醇和水组成，甲醇和水的体积比为85：15，流速为1.0mL/min。检测波长为245nm。
实验结果见图11中D(保留时间为6.366min)。根据所述上清液的峰面积和步骤(1)的标准曲线，计算得到上清液中CDN1163浓度为9.604μg/ml，进一步得到载药脂肪体中CDN 1163浓度为960.4μg/ml。
上述结果表明，实施例1制备的脂肪体可以作为药物载体。
工业应用
以本发明公开的方法制备的脂肪体、人工脂滴和人工脂蛋白，可作为药物载体，进而完成各种生物学和医学的目标，具有重要的应用价值。

Claims (26)

  1. 一种制备由中性脂质和单层磷脂膜组成的脂肪体的方法，包括下述步骤：
    a1)将磷脂和中性脂质在缓冲液中进行涡旋以实现两者的反应，然后进行离心，收集上层液相，从上层液相中分离得到脂肪体。
  2. 如权利要求1所述的方法，其特征在于：所述“从上层液相中分离得到脂肪体”包括如下步骤：
    a2)将所述上层液相进行两次以上的纯化；每次纯化的方法为：将上层液相与所述缓冲液混匀，然后使其分层，收集上层液相；
    a3)将步骤a2)得到的上层液相与所述缓冲液混合，然后使其分层，收集下层液相，其中含有脂肪体。
  3. 如权利要求1或2所述的方法，其特征在于：所述缓冲液为缓冲液B所述缓冲液B的溶质及其在缓冲液中浓度为：15mM～25mM HEPES，80mM～120mM KCl，1.5～2.5mM的MgCl2；溶剂为去离子水；pH为7.2～7.6。
  4. 如权利要求3所述的方法，其特征在于：所述缓冲液B的溶质及其在缓冲液中浓度为：20mM HEPES，100mM KCl，2mM的MgCl2；溶剂为去离子水；pH为7.4。
  5. 如权利要求1至4任一所述的方法，其特征在于：所述步骤a1)中，所述涡旋的参数为：总时长为3～5min；所述步骤a1)中，所述离心的参数为：18000～22000g、3～7min。
  6. 如权利要求5所述的方法，其特征在于：所述涡旋的参数为：总时长4min；所述离心的参数为：20000g、5min。
  7. 如权利要求1至6中任一所述的方法，其特征在于：所述步骤a2)中，所述“上层液相进行两次以上的纯化”中的次数以上层液相与所述缓冲液混匀，分层后无沉淀为准。
  8. 如权利要求1至7中任一所述的方法，其特征在于：所述步骤a2)中，所述“使其分层”是通过离心实现的，所述离心的参数为：18000～22000g、3～7min。
  9. 如权利要求8所述的方法，其特征在于：所述离心的参数为：20000g、5min。
  10. 如权利要求1至9中任一所述的方法，其特征在于：所述步骤a3)中，所述“使其分层”是通过离心实现的，所述离心的参数为：800～1200g、3～7min。
  11. 如权利要求10所述的方法，其特征在于：所述离心的参数为：1000g、5min。
  12. 如权利要求1至11中任一所述的方法，其特征在于：所述磷脂为b1)、b2)或b3)：
    b1)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱；
    b2)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-(9Z- 十八碳烯酰基)-sn-甘油-3磷酸乙醇胺；
    b3)1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱；
    所述中性脂质为c1)或c2)：c1)甘油三酯；c2)胆固醇油酸酯和甘油三酯。
  13. 如权利要求12所述的方法，其特征在于：
    所述b2)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸乙醇胺的质量比为1:0.01～2；
    所述b3)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱的质量比为1:0.01～2；
    所述c2)中，胆固醇油酸酯和甘油三酯的质量比为1～5：1。
  14. 如权利要求13所述的方法，其特征在于：
    所述b2)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸乙醇胺的质量比为2:1、1:1或1:2；
    所述b3)中，1，2-二-(9Z-十八碳烯酰基)-sn-甘油-3磷酸胆碱和1，2-二-十八碳烷酰基-sn-甘油-3-磷酸胆碱的质量比为2:1、1:1或1:2；
    所述c2)中，胆固醇油酸酯和甘油三酯的质量比为5:1、4:1、3:1或2:1。
  15. 如权利要求1至14中任一所述的方法，其特征在于：所述磷脂和所述中性脂质的质量比为(d1)至(d6)中的任一种：(d1)0.25～3:5；(d2)3:5；(d3)2:5；(d4)1:5；(d5)1:10；(d6)1:20。
  16. 用权利要求1-15任一所述方法制备的脂肪体。
  17. 一种制备人工脂滴的方法，包括在权利要求16所述脂肪体上招募一种或几种固有蛋白和/或功能蛋白，获得人工脂滴。
  18. 如权利要求17所述的方法，其特征在于：所述固有蛋白为MLDS蛋白、MDT-28蛋白或Perilipin-2蛋白；所述功能蛋白为ATGL；
    所述MLDS蛋白为如下e1)或e2)：e1)氨基酸序列如序列表中序列2所示的蛋白质；e2)将e1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与MLDS蛋白具有相同功能的蛋白质。
    所述MDT-28蛋白为如下f1)或f2)：f1)氨基酸序列如序列表中序列4所示的蛋白质；f2)将f1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与MDT-28蛋白具有相同功能的蛋白质。
    所述Perilipin-2蛋白为如下g1)或g2)：g1)氨基酸序列如序列表中序列6所示的蛋白质；g2)将g1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与Perilipin-2蛋白具有相同功能的蛋白质。
    所述ATGL为如下h1)或h2)：h1)氨基酸序列如序列表中序列8所示的蛋白质；h2)将h1)所示的蛋白质经过1至10个氨基酸残基的取代和/或缺失和/或添加得到的与ATGL具有相同功能的蛋白质。
  19. 用权利要求17或18所述方法制备的人工脂滴。
  20. 一种制备人工脂蛋白的方法，包括在权利要求16所述脂肪体上招募一种或几种载脂蛋白，获得人工脂蛋白。
  21. 如权利要求20所述的方法，其特征在于：所述载脂蛋白为Apo A-I。
  22. 用权利要求20或21所述方法制备的人工脂蛋白。
  23. (j1)至(j6)中的任一种：
    (j1)权利要求16所述脂肪体在制备人工脂滴和/或人工脂蛋白和/或药物载体中的应用；
    (j2)权利要求19所述人工脂滴在制备药物载体中的应用；
    (j3)权利要求19所述人工脂滴在制备药物中的应用；
    (j4)权利要求22所述人工脂蛋白在制备药物载体中的应用；
    (j5)权利要求22所述人工脂蛋白在制备药物中的应用；
    (j6)权利要求16所述脂肪体作为药物载体的应用。
  24. 一种药物，其活性成分为负载有具有药用功能的化合物的脂肪体；所述脂肪体为权利要求16所述的脂肪体。
  25. 如权利要求24所述的药物，其特征在于：所述药物为脂溶性药物。
  26. 如权利要求24或25所述的药物，其特征在于：所述药物为治疗II型糖尿病和/或代谢紊乱的药物或候选药物。
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