CN101491237A - Use of tetrahydropyridines in cell, tissue, organ cryopreservation - Google Patents
Use of tetrahydropyridines in cell, tissue, organ cryopreservation Download PDFInfo
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- CN101491237A CN101491237A CNA2009100146318A CN200910014631A CN101491237A CN 101491237 A CN101491237 A CN 101491237A CN A2009100146318 A CNA2009100146318 A CN A2009100146318A CN 200910014631 A CN200910014631 A CN 200910014631A CN 101491237 A CN101491237 A CN 101491237A
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- 210000000056 organ Anatomy 0.000 title claims abstract description 34
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 7
- 125000004853 tetrahydropyridinyl group Chemical class N1(CCCC=C1)* 0.000 title 1
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- WQXNXVUDBPYKBA-YFKPBYRVSA-N ectoine Chemical compound CC1=[NH+][C@H](C([O-])=O)CCN1 WQXNXVUDBPYKBA-YFKPBYRVSA-N 0.000 claims abstract description 14
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- FINHMKGKINIASC-UHFFFAOYSA-N Tetramethylpyrazine Chemical compound CC1=NC(C)=C(C)N=C1C FINHMKGKINIASC-UHFFFAOYSA-N 0.000 claims description 4
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Abstract
本发明公开了四氢嘧啶的一种新用途，即四氢嘧啶在冷冻保存细胞、组织、器官中的应用，其有效浓度为0.1g/L－100g/L，优选浓度为0.5g/L－20g/L。使用时可单独使用，也可与一些常规的用于细胞、组织、器官冷冻保存的化学试剂联合使用。本申请的发明人通过试验证明：四氢嘧啶可以帮助细胞抵抗冷冻条件，提高细胞、组织、器官冻存的保护效果。与二甲基亚砜联合使用可以减少其用量降低其对细胞的毒性，提高冻存效果。The invention discloses a new application of ectoine, that is, the application of ectoine in cryopreservation of cells, tissues and organs. The effective concentration is 0.1g/L-100g/L, and the preferred concentration is 0.5g/L- 20g/L. It can be used alone or in combination with some conventional chemical reagents for cryopreservation of cells, tissues and organs. The inventors of the present application have proved through experiments that ectoine can help cells resist freezing conditions and improve the protective effect of cells, tissues and organs on cryopreservation. Combined use with dimethyl sulfoxide can reduce its dosage, reduce its toxicity to cells, and improve the cryopreservation effect.
Description
Technical field
The present invention relates to the application of tetrahydropyrimidine in freezing preservation cell, tissue, organ.
Background technology
In Bioexperiment and disease treatment, often need carry out freezing preservation, in the especially organ transplant process, to the frozen quality that helps guaranteeing donor organ of organ to microorganism and zooblast, tissue, organ.But when low temperature is frozen in the cell and the water in the external environment can form ice crystal, can cause mechanical damage, electrolyte rising, osmotic pressure change, dehydration, PH change, albuminous degeneration etc. take place in the cell, can cause cell death.Frozen protectant commonly used at present is a dimethyl sulfoxide (DMSO); can reduce the cell freezing point, reduce the formation of ice crystal, alleviate the radical pair primary cellular defect; change the permeability of biomembrane to electrolyte, medicine, poisonous substance and metabolite, but dimethyl sulfoxide (DMSO) at normal temperatures pair cell stronger toxic action is arranged.Cause protein denaturation with the protein hydrophobic grouping B that has an effect.Therefore, the consumption that should reduce dimethyl sulfoxide (DMSO) in the frozen process as much as possible reduces the toxicity of its pair cell.
Organ transplant is the major progress of 20th century physianthropy science, and at present, the whole world has surpassed 700,000 examples (inferior) at the transplanting sum in the field of kidney, liver, heart, pancreas and bone-marrow transplantation, and the longest organ transplant recipients of surviving is nearly half a century.Organ transplant is the unique effective radical cure means of the whole organ failure in latter stage of treatment.Any clinical organ transplant at first will have high-quality donor, and this is the prerequisite and the basic guarantee of organ transplant success.
Organ preservative fluid at present commonly used have UW liquid, IGL preserve liquid, Celsior liquid, HTK liquid, SMO preserve liquid, Collins liquid, HC-A liquid, WMO-1 solution, HCA-II preserve liquid, LYPS liquid, STH-1, STH-2 liquid,, Euro-Collins liquid.Its cardinal principle is: reduce because low temperature is preserved the edema that causes; Prevent the acidization of cell; Prevent the expansion in space between cells in the irrigating solution preservation process; Prevent the damage of oxygen radical in the refilling process; The substrate of regeneration high energy phosphate compound is provided.Therefore contain certain impermeable materials, free radical scavenger, cell membrane stability agent and other mitochondrion protecting agents more, improve the material that organ is supported.
1,4,5,6-tetrahydrochysene-2-methyl-4-pyrimidine carboxylic is commonly called as tetrahydropyrimidine (Ectoine), is a kind of novel amino of finding in 1985, be that many salt-durable microbes are to keep the osmotic pressure balance and a kind of compatible solute of producing in cell, have high water soluble.Tetrahydropyrimidine can be stablized the hydration layer of native protein; large biological molecule such as protective enzyme, DNA and membrane structure; help various adverse circumstances such as cell resistance is freezing, arid, high temperature, high salt, radiation; go the commercial use of exploitation at present to concentrate on cosmetics; patent documentation WO0219978 discloses its application in mouth care, and patent documentation DE102004016129 discloses its application in skin nursing and prevention from suffering from the diseases.At present do not find that as yet tetrahydropyrimidine is used for the correlative study and the report of cell, tissue, the frozen protection of organ.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of new purposes of tetrahydropyrimidine.
The present invention comes down to the application of tetrahydropyrimidine in freezing preservation cell, tissue, organ.
The valid density of tetrahydropyrimidine is 0.1g/L-100g/L, and preferred concentration is 0.5g/L-20g/L.
Tetrahydropyrimidine can use separately when freezing preservation cell, tissue, organ, also can unite use with the chemical reagent that is used for cell, tissue, the freezing preservation of organ of some routines.These chemical reagent comprise following a few class:
1, dimethyl sulfoxide (DMSO), glycerine, seralbumin: tetrahydropyrimidine with wherein any or appoint several combinatorial associations to use, can improve the frozen protection effect of cell and tissue.
2, polyethylene glycol, lactobionic acid, kapok sugar, HES, phosphate buffer, histidine buffering liquid, reduced glutathione, perfluorocarbon, Trimetazidine, tryptophan, α-Tong Wuersuan and ligustrazine, magnesium sulfate, potassium dihydrogen phosphate: tetrahydropyrimidine with wherein any or appoint several combinatorial associations to use, can improve the frozen protection effect of organ.
Described cell, tissue, organ comprise animal (comprising the people) cell, tissue, organ, also can be microbial cells.
The present inventor is by evidence: tetrahydropyrimidine can help the cell resistance freezing conditions, improves cell, tissue, the frozen protection effect of organ.Unite use with dimethyl sulfoxide (DMSO) and can reduce the toxicity that its consumption reduces its pair cell, improve frozen effect.
Embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1: tetrahydropyrimidine is to the frozen influence of porcine hepatocyte low temperature
Healthy 1-7 age in days Chinese experimental type miniature pig, adopt the external two step collagenase perfusion method isolating hepatocytes of improvement Seglen, the double layer disinfecting filtered through gauze, 50g then, centrifugal 3 times of 3min obtains liver cell, calculates liver cell quantity with blood cell counting plate under the Olympus inverted phase contrast microscope, 4g/L trypan blue exclusion method meter survival rate surpasses 85% the experiment that is used for.
Cell culture fluid is the L-15 culture fluid, be added with 10ml/L FBS, hydrocortisone 0.1 μ g/L, insulin 100 μ g/L, penicillin 100kU/L and streptomycin 100mg/L, control group adds 50mL/L DMSO, and experimental group adds 50mL/L DMSO and 100mL/L tetrahydropyrimidine.
Divide the frozen pipe of packing into then, at room temperature place 15min, place 4 ℃ of 20min, reduce to-20 ℃ with-1 ℃/min, preserve 30min, reduce to the liver cell after frozen of taking-up behind-80 ℃ of long preservation .30d and the 60d with-1 ℃/min again, place 37 ℃ of water-bath 1-2min fast, slowly dilution adds the about 30mL of culture fluid altogether then, 4 ℃, 30g, centrifugal 3min, abandon supernatant, again be suspended in the culture fluid, calculate cell survival rate (seeing Table 1), and calculate adherent rate.Observe the liver cell form down in the Olympus inverted phase contrast microscope.
Table 1: liver cell survival rate and adherent rate (%) behind the cryopreservation resuscitation
* compare P＜0.01 with control group
The hepatic cell growth situation of control group and recovery back inoculated and cultured of observing under inverted microscope finds that contrast liver cell form is relatively poor, does not see binucleate cell, experimental group group hepatic cell growth is vigorous, the adherent rate height, and cell membrane is complete, the kytoplasm endoparticle is abundant, visible more binucleate cell, and kernel is clear.
Embodiment 2: tetrahydropyrimidine is to the frozen influence of human liver cell low temperature
The separation of human liver cell according to literature method carry out (Wang Yuming, Chen Guozhi, Dong Jiahong, etc. the external perfusion method and the application thereof of isolating hepatocytes.China's experimental surgery magazine, 1994,11 (supplementary issues): 45-46)).Isolated cells is through trypan blue dyeing, counting.It is 1 * 10 that liver cell is transferred to concentration
7-2 * 10
7/ ml branch is packed into, and each 0.7ml is standby for frozen pipe, and control group adds 50mL/L DMSO, and experimental group adds 50mL/L DMSO and 150mL/L tetrahydropyrimidine.Earlier with frozen pipe at 0 ℃ of frozen 30min down, ℃ following frozen 1h then-30, again-80 ℃ down frozen 4h move at last in the liquid nitrogen container, preserves after 1 month and recovers.Calculate survival rate and 24h adherent rate (the results are shown in Table 2), the back is observed under inverted phase contrast microscope and is cultivated hepatocellular growing state and metamorphosis.The supernatant of collecting detects its albumin content (the results are shown in Table 4) with automatic clinical chemistry analyzer, human liver cell after the recovery goes supernatant to wash with the PBS buffer solution after cultivating 3d, add the DMEM culture fluid that contains 4mmol/ml ammonium chloride, serum-free then, after adding ammonium chloride 2,6,12,24h specimen taken respectively, on Biochemical Analyzer, detect urea content (the results are shown in Table 3) in the supernatant.
Table 2: recovery hepatocellular survival rate in back and 24h adherent rate (%)
Table 3: synthetic (mmol/l) of recovery back liver cell urea
Table 4: recovery back hepatocyte cultures supernatant albumin content (g/l)
* compare P＜0.01 with control group
Morphological change under the inverted phase contrast microscope of recovery back, it is fast that experimental group liver cell integrality is recovered, and hepatic cell growth is vigorous, and cell membrane is complete, and the particle of enriching is arranged in the endochylema, and kernel is clear.
Embodiment 3: tetrahydropyrimidine is to the autoplastic influence of rat ovary
Select the female sd inbred rats of not mating for use, the transvaginal smear is looked into cast-off cells and is confirmed the oestrous cycle clocklike.Be divided into 2 groups at random, every group each 10, control group is the frozen back of a DMSO OVARIAN AUTOTRANSPOSITION group, 1 week of stored frozen after the excision bilateral ovaries, and then row is from the body subcutaneous transplantation; Experimental group is the frozen back of a tetrahydropyrimidine OVARIAN AUTOTRANSPOSITION group, 1 week of stored frozen after the excision bilateral ovaries, and then row is from the body subcutaneous transplantation.
In postoperative row vaginal smear the 2nd day every day, observe the variation of vaginal exfoliated.Oestrous cycle variation person occurs, carry out by stages according to the type of cast-off cells; The oestrous cycle person do not occur, light microscopic is the shared ratio of counting keratinocyte down, and＞50% have been considered as estrogen action; Put to death animal after 4 months, size, blood fortune and the peripheral vessels net of observing graft form situation.
None oestrous cycle variation clocklike only appears in control group, just continues no angling state but 8 keratinocyte ratios surpass 50%, 2.6 of experimental group clocklike the oestrous cycle change, the keratinocyte ratio in every visual field surpasses 50% under 3 light microscopics, above-mentioned 9 rats think that estrogen action is arranged.Control group has 1 rat ovarian atrophy to occur in addition, surperficial canescence, and rete vasculosum is less, and the experimental group graft obviously increases, and the peripheral vessels net is abundant, surperficial pinkiness.
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