WO2022242533A1 - 用于清除碎石取石术后残留结石碎块的凝胶组合物 - Google Patents

用于清除碎石取石术后残留结石碎块的凝胶组合物 Download PDF

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WO2022242533A1
WO2022242533A1 PCT/CN2022/092370 CN2022092370W WO2022242533A1 WO 2022242533 A1 WO2022242533 A1 WO 2022242533A1 CN 2022092370 W CN2022092370 W CN 2022092370W WO 2022242533 A1 WO2022242533 A1 WO 2022242533A1
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component
concentration
gel
stone
gel composition
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French (fr)
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周晓晨
匡仁锐
习海波
余月
邓君
陈玉军
陈鑫朋
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/046—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/04—Drugs for disorders of the urinary system for urolithiasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/442—Colorants, dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures

Definitions

  • the invention relates to a gel composition for improving the operation efficiency of calculus and lithotripsy in the urinary system and assisting in removing fragments of calculus in the kidney collecting system.
  • RIRS lithotripsy energy platforms compatible with RIRS
  • the level of surgical operation and the improvement of the understanding of key technologies of RIRS the surgical indications of RIRS are constantly expanding.
  • Some researchers have reported that RIRS can also be safely and effectively treated for stones larger than 2.0 cm, even staghorn stones, complex stones (including solitary kidney stones, horseshoe kidneys, and spinal deformities) and children with urinary stones.
  • RIRS has been widely used in my country and has become an important surgical method for the treatment of urinary stones.
  • popcorn-like lithotripsy pop-dusting refers to placing the tip of the laser fiber into the calices where crushed stones gather
  • the laser is continuously excited.
  • the gravel in the calices will randomly approach the end of the optical fiber and be subjected to the gravel action of the laser, resulting in continuous fragmentation and disintegration.
  • Dustification the fine stone fragments and dust (usually defined as stone fragments ⁇ 2-3mm) produced by popcorn-like gravel need to be discharged by the patient himself by means of increasing drinking water, drugs, and special positions.
  • the main technical solutions adopted in the present invention include:
  • a gel composition for removing residual stone fragments after lithotripsy comprising A component and B component;
  • plasminogen is added to component A and/or component B.
  • non-cytotoxic dyes are added to component A and/or component B. Stains are primarily for visualization and observation.
  • the dyeing agent, Ca 2+ , and plasminogen can be added to component A or component B alone, or a part can be added to both component A and component B.
  • Component A is the gel main agent
  • B component is the gel catalyst. After mixing A and B, a gel with wrapping and adhesion capabilities can be formed.
  • said fibrinogen and fibrin stabilizing factor are derived from human or animal blood or blood products.
  • the fibrinogen and fibrin stabilizing factor in component A can also be derived from human blood, pig blood, cow blood or sheep blood.
  • component A also includes physiological saline; fibrinogen and fibrin stabilizing factor are diluted in physiological saline.
  • component A is composed of fibrinogen and fibrin stabilizing factor, methylene blue and physiological saline; wherein, the concentration of methylene blue is 20.0 ⁇ g/ml-60.0 ⁇ g/ml.
  • the thrombin and plasminogen are derived from human or animal blood or blood products.
  • the thrombin in component B can be one of human thrombin, porcine thrombin, bovine thrombin or sheep thrombin, as long as it can be combined with the source of fibrinogen and fibrin stabilizing factor in component A Correspondence is to meet the use requirements. Where the source is appropriate, thrombin drives the transformation of fibrinogen into a gel.
  • component B also includes physiological saline; thrombin is diluted in physiological saline.
  • component B is composed of thrombin, Ca 2+ , plasminogen and physiological saline; wherein, the concentration of plasminogen is 0.1mg/ml-1mg/ml ml.
  • plasminogen can come from any one of human blood, pig blood, cow blood or sheep blood, but it should correspond to the source of fibrinogen and fibrin stabilizing factor in component A.
  • the gel composition of the present invention is used for efficiently removing fine stone fragments in the kidney, ureter or bladder.
  • the two groups A and B are divided into separate preparations or sub-packages.
  • inject the two into the kidney, ureter or bladder at the same time through a device with a spray function (inject or inject immediately after mixing well) After natural mixing) to form a gel that wraps and adheres to the stone, the gel that adheres to and wraps the stone (via the soft mirror sheath) is taken out by means of negative pressure suction and/or a stone extraction basket.
  • the gel composition of the present invention has added a non-toxic dye to human cells, which helps the gel composition to be injected into the designated area under direct vision, and has good visibility to facilitate stone removal operate.
  • the color of the gel is related to the concentration of dyes such as methylene blue. Users can adjust the color of the gel by adjusting the concentration of the dye according to actual needs.
  • the gel composition of the present invention the fibrin gel formed by it, its softness, plasticity and flexibility and the concentration of fibrinogen and fibrin stabilizing factor in A component (main glue), and B
  • the concentration of thrombin and Ca 2+ in the component (catalyst) is related, so it can be adjusted according to the needs or the specific equipment for removing residual stone fragments during actual use.
  • the present invention also adds plasminogen in A component or B component, makes the fibrin gel that A, B component forms can be in urokinase-normal saline solution or normal people's urine It can be dissolved naturally in the medium, reducing or eliminating the risk of urinary system obstruction caused by the gel.
  • the present invention can cooperate with a variety of urinary system endoscopes to assist in the removal of small stone fragments located in the renal collecting system, ureter and bladder, significantly improve the stone-free rate of urinary stone surgery, and reduce the risk of postoperative stone recurrence in patients. Important clinical promotion value.
  • Figure 2 (A) is a schematic diagram of flexible ureteroscope and pipeline connection; (B) is to inject component A and component B (blue) through the channel of the flexible lens at the same time, aiming at the renal calyx where the stone is located, and then injecting 1ml Operation diagram of saline solution. (C) is the case of gel-coated stones after waiting for 3-5s.
  • Figure 3 shows the ⁇ 1mm (D), ⁇ 2mm (E) and ⁇ 3mm (F) stones screened by natural air-dried and ground human stone specimens with 1mm (A), 2mm (B) and 3mm (C) wire mesh sieves components.
  • Fig. 4 is a photo of the natural dissolution of the gel formed by diluting and undiluted two-component gel in physiological saline in normal human urine and physiological saline.
  • Fig. 5 is a process photo of the method for constructing an isolated pig kidney human calculus model.
  • Fig. 6 shows the gel wrapped with calculus taken out after flexible ureteroscopy in the isolated pig kidney and human calculus model; among them, Fig. 6 (A): through the negative pressure suction of the working channel of the flexible mirror, the gel is guided through the ureter The guide sheath was pulled out of the body; Figure 6(B): the gel surrounding the calculus was sucked out directly through the ureteral introducer sheath with negative pressure; Figure 6(C): the gel enclosing the calculi was grasped by the mesh basket.
  • Fig. 7 is a photo of the gel formed by mixing different final concentrations of methylene blue in component B under white background and light-transmitting state.
  • Fig. 8 is the plain film (A) of the abdomen of a patient with right ureteral calculi; the CT (B) of the lower abdomen after the patient underwent transurethral flexible ureteroscopic lithotripsy using the gel composition of the present invention, no obvious residual stones were found .
  • Figure 9 is a picture of the process of transurethral flexible ureteroscopic lithotripsy for this patient;
  • Figure 9a is a conventional technique to indwell a 12/14Fr ureteral guide sheath, flexible ureteroscope to detect the condition in the kidney, locate the stone, and perform laser stone popcorn powder
  • Figure 9b is a picture of confirming that the stones have been pulverized to less than 2mm fragments;
  • Figure 9c is a picture of rapidly injecting diluted component A and diluted component B into designated areas at the same time;
  • Figure 9d is an observation gel picture of the position;
  • Figure 9e is used for The picture of the process of taking out the colloid from the stone mesh basket;
  • Figure 9f is a picture of the gel composition with a large number of stone fragments taken out during the operation.
  • the basic technical solution of the present invention is: a gel composition for removing residual calculus fragments after lithotripsy, which includes component A and component B prepared or packaged separately; wherein, component A is at least Comprising fibrinogen and fibrin stabilizing factors, the B component contains at least thrombin.
  • component A is at least Comprising fibrinogen and fibrin stabilizing factors
  • the B component contains at least thrombin.
  • Ca 2+ and plasminogen are added to at least one of component A or component B.
  • each component in component A and component B needs to be diluted to a certain extent with physiological saline for use.
  • non-cytotoxic stains such as methylene blue are also added to Component A or Component B.
  • methylene blue can also be replaced with chlorophyll or indocyanine green.
  • the concentration of the stain determines the color of the gel, so the concentration of the stain can be adjusted to meet the visualization needs. If the color is too dark, it is difficult to observe the stone fragments wrapped in gel, and if the color is too light, it is difficult to be visually observed.
  • the concentration in physiological saline is 20.0 ⁇ g/ml-60.0 ⁇ g/ml.
  • the fibrinogen contained in the A component of the present invention, the fibrin stabilizing factor and the thrombin contained in the B component and Ca 2+ can be rapidly formed in the physiological saline and urine environment after being mixed with a certain degree of toughness and plasticity. , Fibrin gel capable of adhering and wrapping stones.
  • the methylene blue contained in component A as a chromogen that has been widely used clinically, can effectively highlight the position of fibrin gel in saline and urine (for easy observation and operation), and does not affect Display of its adhered, encased stones.
  • the plasminogen in the B component can be activated by the thrombin in the catalyst into the plasmin that can dissolve the fibrin gel, but it does not affect the rapid formation of the fibrin gel; the blood in the B component Plasminogen can also be activated by urokinase in urine to form plasmin, which has the activity of hydrolyzing fibrin gel, which helps to accelerate the dissolution and excretion of unintended residual fibrin gel in the body.
  • the softness, plasticity and toughness of the gel are directly related to the "concentration of fibrinogen and fibrin stabilizing factor" in component A and the “concentration of thrombin and Ca 2+ " in component B, so in During use, it can be adjusted according to actual needs or specific equipment for removing residual stone fragments.
  • fibrinogen and fibrin stabilizing factor are derived from human or animal blood or blood products.
  • fibrinogen and fibrin stabilizing factor can also be derived from human blood, pig blood, cow blood or sheep blood.
  • thrombin and plasminogen are derived from human or animal blood or blood products.
  • the thrombin in component B can be one of human thrombin, porcine thrombin, bovine thrombin or sheep thrombin, as long as it can be combined with the source of fibrinogen and fibrin stabilizing factor in component A Correspondence is to meet the use requirements. Where the source is appropriate, thrombin drives the transformation of fibrinogen into a gel.
  • plasminogen can come from any one of human blood, pig blood, cow blood or sheep blood, but it should correspond to the source of fibrinogen and fibrin stabilizing factor in component A.
  • Fibrinogen contained in component A Fibrinogen is the precursor of fibrin, mainly synthesized by liver cells in humans and animals, and is the coagulation factor with the highest content in plasma.
  • the molecular weight of fibrinogen is about 340kDa, and it is a triple globular protein composed of three pairs of polypeptide chains, ⁇ , ⁇ , and ⁇ .
  • Fibrinogen forms fibrin monomers under the action of thrombin, fibrin stabilizing factor and Ca2+ and other coagulation factors, and covalently binds to each other to form fibrin polymers, and its ⁇ chains overlap and covalently cross-link to form stable fibers
  • the protein network forms a gel, and when the concentration of other components is determined, the toughness and plasticity of the gel depend on the concentration of fibrinogen.
  • the gel has low softness, poor plasticity, is difficult to deform, and cannot be sucked out by negative pressure; and if the toughness is low, it is easy to break during the process of being sucked by negative pressure, and cannot effectively "wrap and grab stone fragments".
  • Fibrin stabilization factor contained in component A (main glue): Fibrin stabilization factor, also known as coagulation factor XIII (FXIII), is a glycoprotein synthesized in the bone marrow and liver of humans and animals.
  • the molecular weight of fibrin stabilizing factor is about 340kDa, and it is a tetrameric glycoprotein composed of two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIIIB).
  • Fibrin stabilization factor participates in the formation of thrombin, and can cross-link the ⁇ chain and ⁇ chain of fibrin, which contributes to the rapid formation of fibrin network (generating gel) and resistance to fibrinolysis, and obtains a strong Gel toughness.
  • Methylene blue is an aromatic heterocyclic compound, the chemical name is 3,7-bis(dimethylamino)phenothiazine-5-onium chloride, the chemical formula is C 16 H 18 N 3 ClS, and it is registered in CAS No. 61-73-4, easily soluble in water.
  • Methylene blue is widely used as a chemical indicator, dye, biological stain and clinical drug, and it has also been tried clinically for the treatment of urinary calculi, obliterative vasculitis and neurodermatitis.
  • the aqueous solution of methylene blue is blue in physiological saline, and it can be reduced to a colorless state when it encounters reducing agents such as ammonia water.
  • Thrombin in component B a proteolytic enzyme formed after activation of thrombin prothrombin (coagulation factor II), with a molecular weight of about 37kDa, composed of two peptide chains with molecular weights of 31kDa and 6kDa respectively through disulfide bonds .
  • thrombin catalyzes the formation of fibrin monomers from fibrinogen, and can also activate fibrin stabilization factor (XIII) to become XIIIa, so that fibrin monomers are connected to each other to form water-insoluble fibrin polymers, which are interwoven with each other to form a network. A gel with certain flexibility and plasticity is obtained.
  • thrombin also has the effect of activating plasminogen.
  • Ca 2+ is an indispensable cation in various blood coagulation pathways in the body.
  • Ca 2+ can assist in the activation of factor XI, and activate factor X together with factor VIII and activated factor IX; in the extrinsic coagulation pathway, Ca 2+ activates factor X together with factor III and factor VII ;
  • Ca 2+ can work with factor V and activated factor X to convert fibrinogen into fibrin monomer.
  • Ca 2+ can also assist in the activation of fibrin stabilizing factors, and continue to assist fibrin stabilizing factors in converting soluble fibrin monomers into stable fibrin polymers, so Ca 2+ is mainly used to promote the rapid formation of gels .
  • Plasminogen in component B is the Inactive precursor. Plasminogen directly becomes plasmin through the activation of tissue activator, urine activator (urokinase) and the like. Plasmin is a proteolytic enzyme capable of dissolving fibrin clots (glues). Plasminogen is added to help the gel remaining after removing stone fragments to dissolve naturally after encountering urine, reducing or eliminating the risk of urinary system obstruction.
  • the above-mentioned plasminogen may not be added to the B component (catalyst).
  • FIG. 1A-B normal saline and a certain amount of naturally air-dried and ground human calculus specimens are placed in a vial. Then, mix a certain amount of A component (fibrinogen, fibrin stabilizing factor) and a certain amount of B component (containing thrombin, Ca 2+ and plasminogen) and mix them through a special injection tube. In the normal saline environment of the cillin bottle, a firmer, milky white fibrin gel can be formed within a few seconds (Fig. 1A, B).
  • a component fibrinogen, fibrin stabilizing factor
  • B component containing thrombin, Ca 2+ and plasminogen
  • Component A the concentration of fibrinogen is 2.2 mg/ml; the concentration of fibrin stabilizing factor is 1.6 mg/ml.
  • Component B the thrombin concentration is 20IU/ml, the Ca 2+ concentration is 5mM, and the plasminogen concentration is 1mg/ml.
  • the experimental results showed that the fibrin gel formed by the undiluted AB two-component was relatively tough and could adhere to and wrap stones, but it was difficult to take it out of the body through a ureteral guide sheath with an inner diameter of 12Fr.
  • the fibrin gel formed when diluted 32 times has both good toughness and plasticity, can adhere to and wrap stones, and can be taken out of the body through a ureteral guide sheath with an inner diameter of 12Fr.
  • Fibrin gels formed when diluted 64-fold or higher were not effective in adhering and encapsulating stones.
  • a translucent gel (C) can be formed within 3-5 s; the formed gel can adhere to and wrap stone fragments, and at the same time has ideal plasticity And toughness, it can be easily sucked out of the body through the negative pressure of the 12Fr ureteral guide sheath (the head of the guide sheath does not need to touch the stone, and the distance from the stone deposited at the bottom of the bottle is about 0.5cm) (D), and the stone extraction net basket can also be used Take it out of the body. After the stones and gel were removed, about 0.5 cm thick normal saline remained at the bottom of the bottle (E).
  • Method 1 The AB two-component used is the two-component diluted 32 times in Example 2, which is injected sequentially into the designated liquid environment.
  • Method 2 The AB two-component used is the two-component diluted 32 times in Example 2, mixed and injected into the designated liquid environment within 3 seconds.
  • Method 3 Use 3 infusion tees to inject the 32-fold diluted AB component in Example 2 into the designated area through the working channel of the soft mirror at the same time (at this time component B is added with methylene blue), and then add a small amount of normal saline .
  • the components injected first may be rapidly diluted in the liquid environment, and a gel with expected physical properties cannot be formed.
  • the adult ureter is about 25-35cm long, the male urethra is about 20-22cm long, and the female urethra is about 4-6cm long.
  • the patency of the RIRS operation needs to place the UAS from the external urethral opening to the ureteropelvic junction in order to perform relatively safe lithotripsy for stones in the kidney;
  • the length of the UAS used is generally 46cm (male) and 36cm (female), and the inner diameter is generally 12Fr (about 3.82mm in diameter), so the volume in the UAS lumen is about 5.27ml (male) and 4.12ml (female).
  • the flexible ureteroscope needs to be inserted into the kidney through the UAS.
  • the length of the currently used flexible ureteroscope is generally 60cm, its outer diameter is generally about 8-9Fr (diameter about 2.55-2.87mm), and the inner diameter of the working channel is generally 3.5-4Fr (about 1.11-1.27mm in diameter), so the volume of the working channel of the soft mirror is about 0.58-0.76ml.
  • the volume of the normal adult renal pelvis is about 3-10ml (average 7.5ml), and the volume of the renal calyces is even smaller.
  • This embodiment mainly studies the characteristics that the fibrin gel formed by the gel composition of the present invention can be naturally dissolved in urine and saline environment.
  • the method is as follows: the tough high-density fibrin gel formed by the undiluted AB two-component (this time B component is added with methylene blue) in Example 2, and the 32-fold diluted AB two-component (this time The fibrin gel formed by adding methylene blue to component B) was soaked in normal human urine (37°C).
  • Figure 4(A) is the fibrin gel formed by diluting 32 times
  • Figure 4(B) is the fibrin gel formed without dilution.
  • human urine right
  • all fibrin gels were dissolved after standing in a 37°C water bath for 24 hours (C, D). This shows that fibrin gel can be naturally dissolved in urine.
  • the gel composition of the present invention is applied to isolated pig kidneys to remove pre-implanted human stones, and the stone-free rate is calculated.
  • the experimental method is as follows:
  • the main stone fragments produced during the actual lithotripsy usually include stone fragments and dust in a certain size range, so two kinds of stone fragments with different specifications were used to construct the isolated pig kidney human stone model.
  • the construction method of the isolated pig kidney human calculus model is as follows:
  • the stone removal mode of the experimental group adopt the 32-fold diluted AB double-component in Example 2 (at this time, the B component adds methylene blue), use the injection method shown in Figure 2 in Example 3 (use 3 infusion three-way , inject the A and B components of the fibrin gel at the same time through the working channel of the soft microscope, and then add a small amount of normal saline) into the isolated pig kidney, and use negative pressure suction and stone extraction basket in combination (as shown in Figure 6) .
  • the stone extraction mode of the control group the traditional negative pressure suction and the stone extraction basket were used.
  • the implantation volume of each stone was fixed in the control group and the experimental group. After the operation, the collection system was cut open to collect residual stones, and the stone-free rate was calculated indirectly:
  • Calculus clearance rate (1-mass of residual calculus/mass of implanted calculus)*100%.
  • control group 34.2%, 45.6%, 65.3%
  • the total mass of air-dried calculi implanted in a single kidney is 100mg
  • the AB two-component diluted 8, 16 or 32 times in this embodiment can form a gel in the liquid environment of normal saline, and will not block the working channel of the flexible ureteroscope; It is very convenient to use a guide wire or a stone basket to dredge.
  • the gel formed by the AB double group diluted 8, 16 or 32 times in the liquid environment injected with physiological saline can effectively adhere to and wrap stone fragments of ⁇ 2mm; gel adhesion and
  • the gel-stone complex formed by wrapping the stone has ideal plasticity and toughness; the gel-stone complex can be taken out through the ureteral guide sheath with an inner diameter of 3-4mm by means of a stone extraction basket.
  • the gel composition of the present invention is applied to isolated pig kidneys to remove pre-implanted human stones.
  • the state and characteristics of gel formation may change. Therefore, this embodiment focuses on evaluating the gel two-component adhesive wrap diluted 8, 16 or 32 times under the condition of maintaining the circulation flow rate of 5, 10, 20ml/min normal saline (in line with the perfusion flow rate used in the actual operation). The characteristics of the stone, the shape of the gel-stone complex formed and whether the ureteral introducer sheath is removed.
  • the A component and B component of the gel composition used in this embodiment are the same as in Example 7.
  • the experimental method is basically the same as in Example 7, except that the circulation flow rate of 5, 10, and 20 ml/min of normal saline is kept in the vial containing 10 ml of normal saline and a small amount of ⁇ 2 mm human calculus fragments at the bottom, that is, continuously flowing to A peristaltic pump with a constant flow perfusion function was used in the vial to continuously pour physiological saline into the vial at flow rates of 5, 10, and 20 ml/min.
  • This embodiment is basically the same as Example 8, except that the gel two-components diluted 8 and 10 times (diluted with normal saline) are used (the numerical value of the dilution factor is relative to the undiluted one in Example 7). In terms of stock solution) for experiments.
  • the 10-fold dilution is more convenient for the calculation of dispensing in actual clinical application.
  • the A component and the B component of the gel composition used in this embodiment are basically the same as the gel two components diluted 10 times in Example 9, the difference is that in this embodiment, the B component is added 0, 20, 60, 120, 240 ⁇ g/ml of methylene blue. That is, in the present embodiment, the undiluted stock solution of component A (main glue) and component B (catalyst) is composed as follows:
  • the fibrin glue that is consistent with the principle of the composition of the gel composition of the present invention and has been widely used in surgical wound hemostasis: porcine fibrin sealant (Porcine fibrin sealant, Bioseal Biotech, Guangzhou, China), through After diluting with normal saline and adding methylene blue (methylene blue injection, 111598, 10 mg/ml, Jumpcan, Jiangsu, China), it was applied to patients with renal and ureteral calculi for flexible ureteroscopic lithotripsy.
  • porcine fibrin sealant Porcine fibrin sealant, Bioseal Biotech, Guangzhou, China
  • Surgical method a 12/14Fr ureteral guide sheath was indwelled by conventional techniques, a flexible ureteroscope was used to detect the condition in the kidney and locate the stones, and the stones were pulverized by laser (Fig. 9a). fragments (Fig. 9b).
  • Glue production add 5ml of pig-derived fibrin adhesive component A to 45ml of normal saline; add 5ml of pig-derived fibrin adhesive component B and 0.4ml of methylene blue to 45ml of normal saline.

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Abstract

一种用于提高泌尿系统结石碎石取石手术效率、协助清除肾脏收集系统内结石碎块的凝胶组合物，包括A组分和B组分；A组分含有纤维蛋白原和纤维蛋白稳定因子；B组分含有凝血酶；此外，A组分和/或B组分中添加有Ca 2+、血纤维蛋白溶酶原和染色剂。所述的凝胶组合物在尿液和生理盐水环境中可快速形成具有颜色辨识度的、一定韧性、可塑性、含有血纤维蛋白溶酶原的纤维蛋白凝胶，可粘附、包裹处于尿液和生理盐水环境中的结石碎块，并通过使用负压吸引和/或取石网篮取出体外，尤适于高效清除经碎石取石术后残留的、目前尚无有效方法取出的细小结石碎块，具有良好的生物安全性，实现85％以上的结石清除率，对收集系统和手术器械无任何损伤；此外，纤维蛋白凝胶在尿液中可自然溶解，无导致泌尿系统梗阻的风险。

Description

用于清除碎石取石术后残留结石碎块的凝胶组合物 技术领域
本发明涉及一种用于提高泌尿系统结石碎石取石手术效率、协助清除肾脏收集系统内结石碎块的凝胶组合物。
背景技术
泌尿系结石为泌尿系统良性疾病，但它可能导致尿路梗阻、感染、疼痛，同时伴随诱发尿路上皮恶变的风险，是威胁人类健康的重要疾病。
体外冲击波碎石术(Extracorporeal Shockwave Lithotripsy，ESWL)、输尿管镜碎石术(Ureteroscopic Lithotripsy)和经皮肾镜碎石术(percutaneous nephrolithotomy，PCNL)是目前治疗泌尿系结石的三大治疗手段。软性输尿管镜(Flexible Ureteroscope)诞生于1964年，Marshall教授首先尝试使用软性输尿管镜，经尿道、膀胱逆行至肾脏收集系统进行泌尿系结石手术，称为逆行性肾内手术(Retrograde Intrarenal Surgery，RIRS)；随后从1995年钬激光开始与软镜结合用于上尿路结石、梗阻和肿瘤的治疗，真正开始了输尿管软镜应用的新局面，输尿管软镜被广泛地运用上尿路腔内疾病的诊断与治疗。与传统的硬性输尿管镜相比，软镜可被动弯曲的镜体和可被动弯曲、主动弯曲270°的头端，使其基本上可到达整个集合系统，可操作的空间范围更广。而与PCNL相比，软镜经自然腔道，对肾实质无人为创伤，出血风险显著降低，可探查的范围更广，被认为是治疗大多数小于2cm肾输尿管结石的首选治疗方式。
随着可与RIRS配伍的碎石能量平台的发展、手术操作水平和对RIRS关键技术认识的提高，RIRS的手术适应症在不断拓展。有学者报道，对于2.0cm以上、乃至鹿角形结石、复杂性结石(包括孤立肾结石、马蹄肾、脊柱畸形患者)和小儿尿路结石等，也可安全有效的采用RIRS处理。近10年来，RIRS在我国得到广泛应用，已成为治疗泌尿系结石的重要术式。
目前RIRS碎石取石术的标准操作包括：使用取石网篮等手段将目标结石抓放、移动至便于碎石的单个肾盏(多为中、上盏，盏颈口较小，盏内空间较大、足以充分容纳目标结石)后，进行激光碎石(碎块化和粉末化)操作，随后反复使用套石网篮将较大的碎石屑套出，此过程中检查碎石堆中有无较大的结石，必要时补充碎石；对体积较小而不能套出的结石需行爆米花样碎石(pop-dusting，指的是将激光光纤头端至于碎石聚集的肾盏中较为安全的中央区域，进行激光的持续激发，此时肾盏中的碎石将在水流和激光所产生动能的作用下，随机贴近光纤头端而受到激光的碎石作用，不断发生碎片化和粉尘化)；爆米花样碎石所产生的细小结石碎片和粉尘(通常定义为<2-3mm的结石碎片)，需依靠患者自身通过增加饮水、药物、特殊体位等手段自行排出。因手术器械和手术技术的限制，RIRS术后残留结石的概率不容忽视。研究表明，RIRS术后<3mm结石残留率为10-15％，<2mm结石残留率为16.1％，而<1mm结石残留率则高达86％。
随着人们健康意识的不断提高，超过RIRS所适用结石负荷的高结石负荷患者越来越少。同时得益于RIRS设备的普及化和技术的提高，RIRS在泌尿系结石中的占比呈逐年增长趋势。但如前所述，因技术和器械受限，RIRS术后往往难以避免结石残留，甚至常有较多细小结石碎片和粉末残留、需患者术后自行排石。然而很不幸的是，尽管大部分RIRS术后患者的残留结石可自行排出，残留结石自行排出的时间较长，且仍有约20-30％的患者无法排出结石并发生结石负荷的加重。近期一项研究对148名术后残留结石患者的患者进行了长达2年的随访，残留结石<1mm的平均自行排石时间为9个月，其中18.1％的患者出现结石负荷的增加；残留结石在1-3mm者平均自行排石时间为13.9个月，其中28.6％的患者出现结石负荷的增加。另有一项随访约54个月142例软镜碎石术后残留结石的研究显示，残留结石在1-3mm的自行排石率仅为30.23％。这些调查表明，残留结石自行排出的时间较长，且仍有较多数患者无法排出结石并 发生结石负荷的加重。因此，避免RIRS术后残留结石具有重要意义，而目前尚无效果确切、能够高效取出RIRS术后细小结石碎片的方法或制剂。
发明内容
(一)要解决的技术问题
有鉴于现有技术的问题，本发明提供一种用于清除碎石取石术后残留结石碎块的凝胶组合物，有助于显著改善结石患者RIRS术后的转归，具有重要的科学意义和推广价值。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
一种用于清除碎石取石术后残留结石碎块的凝胶组合物，其包括A组分和B组分；
A组分包含：纤维蛋白原和纤维蛋白稳定因子；
B组分包含：凝血酶；
其中，A组分和/或B组分中添加有Ca 2+。
优选地，A组分和/或B组分中添加有血纤维蛋白溶酶原。
根据本发明的较佳实施例，在A组分和/或B组分中添加有无细胞毒性的染色剂。染色剂主要是为了便于可视化和观察。
所述染色剂、Ca 2+、血纤维蛋白溶酶原既可单独加到A组分中，也可单独加到B组分中，还可在A、B组分中均加入一部分。A组分为凝胶主剂，B组分为凝胶催化剂，A和B混合后可形成具有包裹、粘附能力的凝胶。
根据本发明的较佳实施例，所述染色剂为亚甲蓝、叶绿素或吲哚青绿。
根据本发明的较佳实施例，所述纤维蛋白原和纤维蛋白稳定因子来源于人或动物血液或血液制品。具体地，组分A中的纤维蛋白原和纤维蛋白稳定因子还可来源于人血、猪血、牛血或羊血。
根据本发明的较佳实施例，A组分中，还包含生理盐水；纤维蛋白原和纤维蛋白稳定因子被稀释在生理盐水中。
根据本发明的较佳实施例，A组分是由纤维蛋白原和纤维蛋白稳定因子、亚甲蓝和生理盐水所组成；其中，亚甲蓝的浓度为20.0μg/ml-60.0μg/ml。
根据本发明的较佳实施例，所述凝血酶和血纤维蛋白溶酶原来源于人或动物血液或血液制品。
具体地，B组分中的凝血酶可以是人凝血酶、猪凝血酶、牛凝血酶或羊凝血酶等其中一种，只要能与A组分中的纤维蛋白原和纤维蛋白稳定因子的来源对应即满足使用要求。在来源对应的情况下，凝血酶可促使纤维蛋白原转变成凝胶。
根据本发明的较佳实施例，B组分中，还包含生理盐水；凝血酶被稀释在生理盐水中。
根据本发明的较佳实施例，B组分是由凝血酶、Ca 2+、血纤维蛋白溶酶原和生理盐水所组成；其中，血纤维蛋白溶酶原的浓度0.1mg/ml-1mg/ml。
其中，血纤维蛋白溶酶原可来自于人血、猪血、牛血或羊血中的任一种，但要与A组分中的纤维蛋白原和纤维蛋白稳定因子的来源对应。
本发明的技术效果为：
本发明的凝胶组合物，用于高效取出肾脏、输尿管或膀胱内的细小结石碎片。在使用前A、B两组分为单独配制或分装，在使用时通过具有喷射功能的装置，将二者同时注入肾脏、输尿管或膀胱中的结石所在区域(可混匀后立即注入或注入后自然混匀)，形成包裹、粘附结石的凝胶后，采用负压吸引和/或取石网篮等方式将粘附包裹结石的凝胶(经软镜镜鞘)取出。
本发明的凝胶材料具有以下特点：
(1)本发明凝胶组合物A、B两组分在生理盐水环境中可快速形成 具有一定韧性、可塑性、含有血纤维蛋白溶酶原的纤维蛋白凝胶，所形成的凝胶可粘附、包裹处于生理盐水环境中的细小结石碎块，粘附、包裹结石的凝胶具有足够的柔软度、可塑性和韧度等，可通过使用负压吸引和/或取石网篮取出体外。本发明凝胶组合物尤适于高效清除经碎石取石术后残留的结石碎块，具有良好的生物安全性，实现85％以上的结石清除率，对收集系统和手术器械无任何损伤。本发明可用于泌尿系统结石的结石手术后的细小结石碎片的清除。
(2)本发明的凝胶组合物加入了对人体细胞无毒性的染色剂，该染色剂有助于凝胶组合物可在直视下注入至指定区域，具有很好的可视性便于取石操作。凝胶的颜色深浅与亚甲蓝等染色剂的浓度有关，使用者可根据实际需求通过调节染色剂的浓度调节凝胶色泽的深浅。
(3)本发明的凝胶组合物，其所形成的纤维蛋白凝胶，其柔软度、可塑性和柔韧度与A组分(主体胶)中纤维蛋白原和纤维蛋白稳定因子的浓度、及B组分(催化剂)中凝血酶及Ca 2+的浓度相关，因而可在实际使用时根据需求或具体取出残留结石碎块的器械进行调节。
(4)本发明还在A组分或B组分中加入了血纤维蛋白溶酶原，使A、B组分所形成的纤维蛋白凝胶可在尿激酶-生理盐水溶液或正常人尿液中可自然溶解，减少或消除凝胶导致泌尿系统梗阻的风险。
本发明可配合多种泌尿系统腔内镜、协助取出位于肾脏集合系统、输尿管和膀胱内的细小结石碎块，显著提高泌尿系结石手术的结石清除率，降低患者术后结石复发的风险，具有重要的临床推广价值。
附图说明
图1的A-B为本发明凝胶组合物在生理盐水中形成凝胶的照片；图1C-E为凝胶双组分稀释25-35倍时，形成的半透明凝胶能够粘附包裹结石碎块，并可经12Fr的输尿管导引鞘负压容易地吸出到体外的实验照片。
图2的(A)图为输尿管软镜及管道连接示意图；(B)为将A组分和B组分(蓝色)同时经软镜通道，对准结石所在肾盏快速注入，随后 注入1ml生理盐水的操作图。(C)为等待3-5s后凝胶包裹结石的情况。
图3为将自然风干、研磨的人结石标本用1mm(A)、2mm(B)和3mm(C)金属网筛筛选出≤1mm(D)、≤2mm(E)和≤3mm(F)结石组分。
图4为将稀释和未稀释的凝胶双组分在生理盐水中形成的凝胶在正常人尿液和生理盐水中自然溶解的照片。
图5为构建离体猪肾人结石模型的方法过程照片。
图6为在离体猪肾人结石模型行输尿管软镜取石术后，取出的包裹结石的凝胶；其中，图6(A)：经软镜工作通道负压吸引，将凝胶经输尿管导引鞘牵引出体外；图6(B)：经输尿管导引鞘直接负压吸出包裹结石的凝胶；图6(C)：网篮抓取包裹结石的凝胶。
图7为B组分中混入不同终浓度的亚甲蓝所形成的凝胶在白底和透光状态下的照片。
图8为某右侧输尿管结石患者的腹部平片(A)；使用本发明的凝胶组合物对患者行经尿道输尿管软镜结石碎石术之后的下腹部CT(B)，未见明显结石残留。
图9为对该患者行经尿道输尿管软镜结石碎石术的过程图片；图9a为常规手法留置12/14Fr输尿管导引鞘，输尿管软镜探查肾内情况、定位结石，行激光结石爆米花粉末化的图片；图9b为确认已将结石粉末化至小于2mm碎块的图片；图9c为将稀释的组分A和稀释的组分B同时快速注入指定区域的图片；图9d为观察凝胶位置的图片；图9e为用
Figure PCTCN2022092370-appb-000001
取石网篮取出胶体的过程图片；图9f为术中取出粘附大量结石碎块的凝胶组合物图片。
具体实施方式
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施方式，对本发明作详细描述。
本发明的基本技术方案为：一种用于清除碎石取石术后残留结石碎 块的凝胶组合物，其包括分别配制或分装的A组分和B组分；其中，A组分至少包含纤维蛋白原和纤维蛋白稳定因子，B组分至少包含凝血酶。此外，在A组分或B组分至少其中之一还添加有Ca 2+和血纤维蛋白溶酶原。其中，A组分中的各成分和B组分中的各成分，均需要使用生理盐水稀释到一定程度进行使用。
为了实现可视化，在A组分或B组分中还添加亚甲蓝等无细胞毒性的染色剂。当然，亚甲蓝还可以用叶绿素或吲哚青绿替换。染色剂的浓度决定了凝胶的色泽，因此可通过调节染色剂的浓度以满足可视化需求。颜色过深，难以观察到结石碎块被凝胶包裹的情况，而颜色过浅，则难以采用被直观地观察到。优选地，当亚甲蓝加到B组分时，在生理盐水中的浓度为20.0μg/ml-60.0μg/ml。
在使用时，采用具有喷射功能的装置，将A、B二者同时注入肾脏、输尿管或膀胱中的结石所在区域，A、B量组分相混合后会快速形成具有一定柔软度、可塑性和韧度的纤维蛋白凝胶，这些纤维蛋白凝胶包裹、粘附结石碎块后，再采用负压吸引和/或取石网篮等方式将其取出体外，达到清除结合碎块的目的。
本发明的A组分中所包含的纤维蛋白原、纤维蛋白稳定因子和B组分中所包含的凝血酶和Ca 2+混合后可在生理盐水和尿液环境中快速形成具有一定韧性和可塑性、能够粘附和包裹结石的纤维蛋白凝胶。A组分中所包含的亚甲蓝作为一种已广泛临床应用的显色剂，可有效的在生理盐水和尿液中凸显纤维蛋白凝胶的位置(以便于观察和操作)，且不影响对其粘附、包裹的结石的显示。B组分中的血纤维蛋白溶酶原可被催化剂中的凝血酶激活为能够溶解纤维蛋白凝胶的血纤维蛋白溶酶，但不影响纤维蛋白凝胶的快速形成；B组分中的血纤维蛋白溶酶原也可被尿液中的尿激酶激活，形成具有水解纤维蛋白凝胶活性的血纤维蛋白溶酶，有助于加速意外残留的纤维蛋白凝胶在体内的溶解与排出。
其中，凝胶的柔软度、可塑性和韧度主要A组分中“纤维蛋白原和 纤维蛋白稳定因子的浓度”以及与B组分中“凝血酶和Ca 2+的浓度”直接相关，因此在使用时可根据实际需要或具体取出残留结石碎块的器械进行调节。
优选地，纤维蛋白原和纤维蛋白稳定因子来源于人或动物血液或血液制品。具体地，纤维蛋白原和纤维蛋白稳定因子还可来源于人血、猪血、牛血或羊血。
优选地，凝血酶和血纤维蛋白溶酶原来源于人或动物血液或血液制品。具体地，B组分中的凝血酶可以是人凝血酶、猪凝血酶、牛凝血酶或羊凝血酶等其中一种，只要能与A组分中的纤维蛋白原和纤维蛋白稳定因子的来源对应即满足使用要求。在来源对应的情况下，凝血酶可促使纤维蛋白原转变成凝胶。
具体地，血纤维蛋白溶酶原可来自于人血、猪血、牛血或羊血中的任一种，但要与A组分中的纤维蛋白原和纤维蛋白稳定因子的来源对应。
对本发明凝胶组合物中各成分的功能及方案原理说明如下：
A组分(主体胶)中所包含的纤维蛋白原：纤维蛋白原是纤维蛋白的前体，主要由人和动物体内肝细胞合成，是血浆中含量最高的凝血因子。纤维蛋白原分子量约为340kDa，是由α、β、γ三对多肽链组成的三联球形蛋白质。纤维蛋白原在凝血酶、纤维蛋白稳定因子和Ca2+等凝血因子的作用下形成纤维蛋白单体，并相互共价结合形成纤维蛋白多聚体，其α链交错重叠共价交联形成稳定的纤维蛋白网，形成凝胶状，在其他组分浓度确定的情形下，凝胶的韧性和可塑性取决于纤维蛋白原的浓度。
凝胶若柔软度低、可塑性差，难以变形，无法采用负压吸引出来；而韧度低，则容易在被负压吸引过程破裂，不能有效“包裹和抓取结石碎块”。
A组分(主体胶)中所包含的纤维蛋白稳定因子：纤维蛋白稳定因子又称为凝血因子ⅩⅢ(FⅩⅢ)，是一种在人和动物的骨髓和肝脏中合成的糖蛋白。纤维蛋白稳定因子分子量约为340kDa，是由2条催化A亚单 位(FⅩⅢ-A)和2条载体B亚单位(FⅩⅢB)组成的四聚体糖蛋白。纤维蛋白稳定因子参与凝血酶形成，且可使纤维蛋白的α链与γ链发生交联，有助于纤维蛋白网的快速形成(产生凝胶)和对纤溶作用的抵抗，获得较强的凝胶韧度。
亚甲蓝：亚甲蓝是一种芳香杂环化合物，化学名称为3,7-双(二甲氨基)吩噻嗪-5-翁氯化物，化学式为C 16H 18N 3ClS，CAS登录号为61-73-4，在水中易溶。亚甲蓝作为化学指示剂、染料、生物染色剂和临床药物被广泛使用，在临床上还被试用于治疗尿路结石、闭塞性脉管炎和神经性皮炎等。亚甲蓝的水溶液在生理盐水中呈蓝色，遇氨水等还原剂可被还原成无色状态。
B组分(催化剂)中的凝血酶：凝血酶凝血酶原(凝血因子II)激活后形成的蛋白水解酶，分子量约37kDa，由分子量分别为31kDa和6kDa的两条肽链通过二硫键组成。凝血酶催化纤维蛋白原形成纤维蛋白单体，还可激活纤维蛋白稳定因子(ⅩⅢ)变为ⅩⅢa，使纤维蛋白单体相互连接形成不溶于水的纤维蛋白多聚体，并彼此交织成网，得到具有一定柔韧度和可塑性的凝胶。此外，凝血酶还具有激活血纤维蛋白溶酶原的作用。
B组分(催化剂)中钙离子(Ca 2+)：Ca 2+是体内多种凝血途径中不可缺少的阳离子。在内源性凝血途径中，Ca 2+可协助激活XI因子，和Ⅷ因子、活化的Ⅸ因子一起激活Ⅹ因子；在外源性凝血途径中，Ca 2+和Ⅲ因子、Ⅶ因子一起激活Ⅹ因子；在共同途径中，Ca 2+可以和Ⅴ因子、活化的Ⅹ因子一起，将纤维蛋白原转变为纤维蛋白单体。另外，Ca 2+还可以协助激活纤维蛋白稳定因子，并且继续协助纤维蛋白稳定因子将可溶性纤维蛋白单体转变为稳定的纤维蛋白多聚体，因而Ca 2+主要用于促进凝胶的快速形成。
B组分(催化剂)中血纤维蛋白溶酶原：血纤维蛋白溶酶原/纤溶酶原(plasminogen，或profibrinolysin)，亦称为血浆胰蛋白酶原(plasmatrypsinogen)，是血纤维蛋白溶酶的无活性前体。血纤维蛋白溶 酶原通过组织的激活剂、尿激活剂(尿激酶)等的激活作用，直接成为血纤维蛋白溶酶。血纤维蛋白溶酶是一种能够溶解纤维蛋白凝块(胶)的蛋白水解酶。加入血纤维蛋白溶酶原，有助于取结石碎块后残留的凝胶遇到尿液后自然溶解，降低或消除泌尿系统梗阻的风险。
B组分(催化剂)中也可以不添加上述血纤维蛋白溶酶原。
为了进一步验证本发明的技术效果，以下结合实施例说明。
实施例1
如图1A-B所示：在西林瓶中装入生理盐水和一定量自然风干、研磨的人结石标本。然后，将一定量的A组分(纤维蛋白原、纤维蛋白稳定因子)和一定量的B组分(含凝血酶、Ca 2+和血纤维蛋白溶酶原)混匀，并经专用注胶管向西林瓶的生理盐水环境中，可在数秒内形成质地较为坚韧的、乳白色的纤维蛋白凝胶(图1A、B)。
在该西林瓶中，纤维蛋白原(2.2mg/ml)、纤维蛋白稳定因子(1.6mg/ml)、凝血酶(20IU/ml)、Ca 2+(5mM)、血纤维蛋白溶酶原(1mg/ml)。
实施例2
本实施例主要研究稀释倍数对凝胶可塑性和韧性的影响。实验过程如下：
(1)配制A组分(主体胶)和B组分(催化剂)未稀释的原液。
A组分：纤维蛋白原的浓度为2.2mg/ml；纤维蛋白稳定因子的浓度为1.6mg/ml。B组分：凝血酶浓度为20IU/ml，Ca 2+浓度为5mM，血纤维蛋白溶酶原浓度为1mg/ml。
A组分中纤维蛋白原+纤维蛋白稳定因子的总浓度为1.8-3.8mg/ml，B组分中凝血酶浓度为15.0-21.7IU/ml。
(2)用生理盐水、采用大范围的等比稀释方式对A组分和B组分进行稀释。稀释倍数为：1.414、2、2.828、4、5.656、8、11.312、16、22.624、32、45.248、64、90.496、128、181.047和256。
(3)在30ml的西林瓶中注入10mL生理盐水和一定量的自然风干、研磨的人结石标本。
(4)将稀释后的A组分和B组分同时注入到西林瓶中以形成凝胶，并采用内径12Fr的输尿管导引鞘对凝胶进行负压吸附实验。
实验结果显示：未稀释的AB双组分形成的纤维蛋白凝胶质地较为坚韧，可粘附、包裹结石，但难以经内径12Fr的输尿管导引鞘取出体外。而稀释32倍时形成的纤维蛋白凝胶兼顾良好的韧性和可塑性，可粘附、包裹结石，并可经内径12Fr的输尿管导引鞘取出体外。稀释64倍或更高倍数时形成的纤维蛋白凝胶无法有效的粘附和包裹结石。
(5)采用小范围等差稀释的方式，将A组分和B组分用生理盐水分别稀释20、25、30、35、40、45倍，将稀释后的A组分和B组分同时注入到西林瓶中以形成凝胶，并采用内径12Fr的输尿管导引鞘(ureter access sheath,UAS)对凝胶进行负压吸附实验。
实验结果如图1C-E所示：稀释25-35倍时，可在3-5s内形成半透明凝胶(C)；形成的凝胶能够粘附包裹结石碎块，并同时具有理想的可塑性和韧性，可经12Fr的输尿管导引鞘(导引鞘头端无需接触结石，距离沉积在瓶底的结石约0.5cm)负压容易地吸出到体外(D)，也可以采用取石网篮抓取到体外。结石与凝胶取尽后，仍可见厚约0.5cm的生理盐水残留于瓶底(E)。
实施例3
本实施例主要研究注入方式对凝胶取石性能的影响。注入方式如下：
方式一：使用的AB双组分为实施例2中稀释32倍的双组分，以先后顺序注入的方式注入指定液态环境中。
方式二：使用的AB双组分为实施例2中稀释32倍的双组分，混匀后在3s内注入指定液态环境中。
方式三：用3个输液三通，经软镜工作通道同时向指定区域注入实施例2中稀释32倍的AB双组分(此时B组分添加了亚甲蓝)，随后追 加少量生理盐水。
实验结果表明：
若采用先后注入的方式注入液态环境中，则可能导致先注射的组分在液态环境中被快速稀释，而无法形成具备预期物理性质的凝胶。
虽然A、B组分混匀后需3-5秒左右才能形成凝胶。但是若在A、B组分混匀后3s内注入到指定区域，在实际应用中容易出现堵塞注射管腔的问题。成人输尿管全长约25-35cm，男性尿道长约20-22cm，女性尿道长约4-6cm。RIRS手术通畅需将UAS从尿道外口放至肾盂输尿管连接处，以便对肾内的结石进行相对安全的碎石取石操作；由于男女解剖差异和男性尿道外口至尿道内口距离的可变性，所使用的UAS长度一般为46cm(男)和36cm(女)，内径一般为12Fr(直径约3.82mm)，因此UAS管腔内的容积大约为5.27ml(男)和4.12ml(女)。输尿管软镜需经UAS探入肾内，目前所使用的输尿管软镜长度一般为60cm，其外直径一般约为8-9Fr(直径约2.55-2.87mm)，工作通道的内径一般为3.5-4Fr(直径约1.11-1.27mm)，因此软镜工作通道的容积大约是0.58-0.76ml。正常成人肾盂的容积约为3-10ml(平均7.5ml)，肾盏的容积更小。若将AB组分混匀后经UAS管腔注入到肾盂内，则将有相对较多的凝胶残留在管腔内(男：5.27ml，女：4.12ml)，需补充注射更多的生理盐水以将残留凝胶冲出管腔内来避免堵管，而UAS管腔过大，也存在生理盐水无法充分冲出的可能。因此，经UAS管腔注入凝胶的方法并不可取。若在软镜工作通道中置入一根内径2Fr、外径3Fr的微导管来实现凝胶双组分的肾内注射，虽然避免了凝胶堵塞软镜工作通道的可能，但增加了手术成本，也因需反复进出导管而增加了手术时间。
如图2所示，使用3个输液三通，一对一连接一个注射器(最外端的注射器装生理盐水、中间注射器装A组分、下方注射器装蓝色的B组分)，输液三通串接起来，末端连接到软镜工作通道。借此，直接经软镜工作通道同时注入纤维蛋白凝胶的A、B组分，随后追加少量生理盐水 (超过软镜工作通道的容积：0.58-0.76ml)，即可将纤维蛋白凝胶的AB双组分注入指定区域，并在生理盐水的液态环境中形成凝胶，同时并不会堵塞软镜工作通道；即便不追加生理盐水冲管，也可很方便的使用导丝或取石网篮疏通。
其中，图2的(A)图为输尿管软镜及管道连接示意图。(B)图为将A组分和B组分(蓝色)同时经软镜通道，对准结石所在肾盏快速注入，随后注入1ml生理盐水，等待3-5s，观察凝胶包裹结石的情况(C)。
实施例4
本实施例主要研究本发明的凝胶组合物配合12Fr的UAS能够取出的结石碎块的大小范围。实验方法如下：
(1)将本单位收集的临床收集的泌尿系结石标本(一水草酸钙和二水草酸钙为主要成分)进行研磨和自然风干处理，随后采用孔径分别为1mm、2mm和3mm的金属网筛，分别筛选出≤1mm、≤2mm和≤3mm的结石组分(图3)。
(2)使用实施例2中稀释32倍的凝胶双组分进行取石试验。
结果显示，被稀释的纤维蛋白凝胶所包裹的≤1mm和≤2mm的结石组分均可经12Fr的UAS负压吸出或网篮取出；≤3mm的结石组分虽可经网篮取出，但在负压吸出时常发生堵管(表1)。
表1.纤维蛋白凝胶取石(12Fr输尿管导引鞘)
Figure PCTCN2022092370-appb-000002
实施例5
本实施例主要研究本发明的凝胶组合物形成的纤维蛋白凝胶可在尿 液和生理盐水环境中自然溶解的特性。方法如下：将实施例2中未稀释的AB双组分(此时B组分添加亚甲蓝)所形成的质地坚韧的高密度纤维蛋白凝胶，和32倍稀释的AB双组分(此时B组分添加亚甲蓝)所形成的纤维蛋白凝胶分别浸泡在正常人尿液(37℃)中。
结果如图4所示，图4(A)为稀释32倍形成的纤维蛋白凝胶，图4(B)为未稀释形成的纤维蛋白凝胶，放入生理盐水(最左)和5份正常人尿液中(右)置于37℃水浴箱中静置24小时后，所有纤维蛋白凝胶均溶解(C、D)。由此说明，纤维蛋白凝胶均可尿液中自然溶解。
实施例6
本实施例是将本发明的凝胶组合物应用到离体猪肾中，用于取出预先植入的人结石，并计算结石清除率。实验方法如下：
(1)准备两种不同大小的结石碎块(≤1mm和≤2mm)构建离体猪肾人结石模型。
在实际碎石手术过程中主要产生的结石碎片通常包含一定大小范围的结石碎片及粉尘，因此构建离体猪肾人结石模型时使用两种不同规格的结石碎块。
离体猪肾人结石模型的构建方法为：
取新鲜家猪猪肾，在肾盂作一长1cm切口，经切口放入筛好的结石碎块及粉末(图5A)，2-0丝线连续锁边缝合关闭肾盂切口(图5B)。将肾脏及肾盂用2-0丝线间断缝合固定于10mm厚硅胶软垫(图5C)。将准备好的固定于硅胶垫的猪肾置于装有37℃生理盐水的塑料箱中，塑料箱由两个高度可调的升降台支撑，使肾脏、肾盂及输尿管符合正常生理解剖位置，塑料箱的侧面密封固定UAS(图5D)。
(2)设置实验组和对照组的取石模式
实验组的取石模式：采用实施例2中32倍稀释的AB双组分(此时B组分添加亚甲蓝)，使用实施例3中图2所示的注射方法(使用3个输液三通，经软镜工作通道同时注入纤维蛋白凝胶的A、B组分，随后追加 少量生理盐水)注入到离体猪肾中，并联合使用负压吸引和取石网篮(如图6所示)。
对照组的取石模式：采用传统的负压吸引和取石网篮。
为了避免技术差异对实验结果的影响，输尿管软镜操作由具有丰富输尿管软镜操作经验的单一术者实施(>1000例RIRS手术经验)。单个肾脏手术操作时间上限设定为30分钟。
对照组和实验组中每次结石的植入量是固定的。手术结束后，剖开收集系统、收集残留结石，对结石清除率进行间接计算：
结石清除率＝(1-残留结石质量/植入结石质量)*100％。
实验结果如表2所示：
①、对于≤1mm结石者，对照组与实验组的操作时间分别为30:00(分，秒)、30:00、28:40和18:35、13:20、10:15，结石清除率分别为：对照组：34.2％、45.6％、65.3％；实验组：89.2％、91.3％、92.1％。由此可见，实验组结石清除率显著高于对照组，p＝0.0096。
②，对于≤2mm结石者，对照组与实验组的操作时间分别为30:00、30:00、30:00和13:42、16:23、8:28，结石清除率分别为：对照组：15.2％、29.6％、16.3％；实验组：92.9％、85.4％、95.0％。由此可见，实验组结石清除率显著高于对照组，p＝0.0002。
表2：离体猪肾纤维蛋白凝胶取石
Figure PCTCN2022092370-appb-000003
Figure PCTCN2022092370-appb-000004
注：
a：单个肾脏植入风干结石的总质量均为100mg；
b：结石清除率的计算公式为：结石清除率＝(1-残留结石质量/植入结石质量)*100％
实施例7
本实施例所使用的凝胶组合物不含实施例2中的血纤维蛋白溶酶原，A组分(主体胶)和B组分(催化剂)未稀释的原液：A组分：纤维蛋白原和纤维蛋白稳定因子的浓度为3.8mg/ml。B组分：凝血酶浓度为20IU/ml，Ca 2+浓度为5mM。
采用实施例2中步骤(2)的稀释方式对本实施例的原液对A组分和B组分进行稀释。
将本实施例所使用的稀释8、16或32倍的AB双组分按照实施3方式三的方式注入到含有10ml生理盐水、底部装有少量<2mm人结石碎块的西林瓶中。虽然在上述实施例3-6中多得出优选稀释倍数为32倍，但本实施例意在观察更低稀释倍数(更高浓度)所产生的的凝胶是否可能堵塞直径仅1-1.2mm的软镜工作通道，或堵塞后是否能够方便而有效的用现有手术材料(直径约0.8-1mm的导丝，或直径约0.8-1.1mm的取石网篮)或简单方法(直接注射生理盐水)疏通。
结果表明本实施例中稀释8、16或32倍的AB双组分能够在生理盐水的液态环境中形成凝胶，同时并不会堵塞输尿管软镜工作通道；即便不追加生理盐水冲管，也可很方便的使用导丝或取石网篮疏通。此外，本实施例中稀释8、16或32倍的AB双组在注入生理盐水的液态环境中所形成的凝胶，能够有效的粘附和包裹<2mm的结石碎块；凝胶粘附和包裹结石所形成的的凝胶-结石复合体具有理想的可塑性和韧性；凝胶-结石复合体能够采用取石网篮的方式经内径3-4mm的输尿管导引鞘取出。
实施例8
本实施例是将本发明的凝胶组合物应用到离体猪肾中，用于取出预先植入的人结石。在水流不断循环的状态下，凝胶形成状态与特性可能 发生改变。因此本实施例重点评估在保持5、10、20ml/min生理盐水循环流量的情况下(符合实际手术中所使用的灌注流量)，稀释8、16或32倍的凝胶双组分粘附包裹结石的特性，所形成的凝胶结石复合体的性状及其是否输尿管导引鞘取出。
本实施例所使用的凝胶组合物的A组分和B组分同实施例7。
实验方法基本同实施例7，不同之处在于向含有10ml生理盐水、底部装有少量<2mm人结石碎块的西林瓶中，保持5、10、20ml/min的生理盐水循环流量，即持续向西林瓶中用带有恒流灌注功能的蠕动泵，以5、10、20ml/min的流量，向西林瓶中持续灌入生理盐水。
结果表明，在5、10、20ml/min的循环流量下，稀释8倍的凝胶双组分所形成的的凝胶粘附包裹结石的特性最优，所形成的凝胶结石复合体足够坚韧、不易断裂，并可用取石网篮经输尿管导引鞘取出；稀释8倍的凝胶双组分所形成的的凝胶可粘附包裹结石，但经网篮抓取、拖出输尿管导引鞘时，容易发生断裂，导致需要更多次的抓取；稀释32倍的凝胶双组分所形成的的凝胶无法充分粘附包裹结石。
实施例9
本实施例与实施例8基本相同，不同之处在于，使用稀释8和10倍(同样使用生理盐水进行稀释)的凝胶双组分(稀释倍数的数值是相对于实施例7中的未稀释的原液而言)进行实验。稀释10倍在实际临床应用中更便于配药计算。
实验方法同实施例8。
结果表明，在5、10、20ml/min的循环流量下，稀释8与稀释10倍的凝胶双组分所形成的的凝胶粘附包裹结石的特性相同，所形成的凝胶结石复合体足够坚韧、不易断裂，并可用取石网篮经输尿管导引鞘取出。可见，实际临床应用中可以采用稀释8倍、稀释10倍或者在上述两稀释倍数之间的任意值。
实施例10
本实施例所使用的凝胶组合物的A组分和B组分与实施例9中稀释10倍的凝胶双组分相同基本，不同之处在于，本实施例中在B组分中添加0、20、60、120、240μg/ml的亚甲蓝。即本实施例中A组分(主体胶)和B组分(催化剂)未稀释的原液组成如下：
A组分：纤维蛋白原和纤维蛋白稳定因子的浓度为3.8mg/ml。B组分：凝血酶浓度为20IU/ml，Ca 2+浓度为5mM。在用生理盐水稀释10倍的B组分中加入亚甲蓝，使亚甲蓝在B组分中的终浓度为0、20、60、120、240μg/ml。
结果显示，未稀释的纤维蛋白凝胶为乳白色，稀释后的凝胶几乎透明，在生理盐水中较难辨别。在B组分中混入终浓度为20-60μg/ml的亚甲蓝在生理盐水有效的将凝胶凸显为蓝色，且不影响操作视野和其中所包裹的结石的显示(图7)。
实施例11
本实施例是将与本发明的凝胶组合物成分原理一致、已广泛应用于外科创面止血的纤维蛋白胶：猪源纤维蛋白粘合剂(Porcine fibrin sealant，Bioseal Biotech，Guangzhou，China)，通过生理盐水稀释、添加亚甲蓝(亚甲蓝注射液，111598，10mg/ml，Jumpcan，Jiangsu，China)后，应用于肾输尿管结石患者实施输尿管软镜碎石取石术。
患者一般资料：女，53岁，住院号D01280643，因右肾积水于2021-03-08入南昌大学第一附属医院泌尿外科，腹部平片(DX202103081630)示：右侧输尿管结石，大小约0.8*1.8cm(图8a)，患者于2021-03-09行经尿道输尿管软镜输尿管结石碎石术(右)。
手术材料：猪源纤维蛋白粘合剂(Porcine fibrin sealant，Bioseal Biotech，Guangzhou，China)、亚甲蓝(111598，10mg/ml，Jumpcan，Jiangsu，China)、导丝(HWS-035150，
Figure PCTCN2022092370-appb-000005
MEDICAL LLC，Indiana，USA)、12/14Fr输尿管导引鞘(女：FUS-120035，
Figure PCTCN2022092370-appb-000006
MEDICAL LLC，Indiana，USA)、输尿管软镜(USCOPE，PUSEN Medical，Guangdong， China)、取石网篮(NTSE-022115-UDH，
Figure PCTCN2022092370-appb-000007
MEDICAL LLC，Indiana，USA)、输尿管支架管(
Figure PCTCN2022092370-appb-000008
Firm，UFI-626-R，
Figure PCTCN2022092370-appb-000009
MEDICAL LLC，Indiana，USA)、输液三通(BD Connecta，394605，BD Medical，Helsingborg，Sweden)、钬激光光纤(SlimLine SIS 200 Laser Fiber，Lumenis，)、钬激光主机(versapulse powersuite 100W Holmium Laser System，Lumenis，Yokneam，Israel)，高压水泵(医用加压器RXJ-I，Tonglu Rex Medical Instrument Co.,Ltd.，Hang Zhou，China)。
手术方法：常规手法留置12/14Fr输尿管导引鞘，输尿管软镜探查肾内情况、定位结石，行激光结石爆米花粉末化(图9a)，粉末化完毕后确认已将结石粉末化至小于2mm碎块(图9b)。制胶:5ml猪源纤维蛋白粘合剂组分A加入45ml生理盐水；5ml猪源纤维蛋白粘合剂组分B和0.4ml亚甲蓝加入45ml生理盐水。准备注胶系统：3个三通管的侧方入水口连接3个20ml注射器，从远端到近端分别装有生理盐水、稀释而蓝染的组分B和稀释的组分A。撤去普通进水通道，连接注胶系统。将稀释的组分A和稀释的组分B同时快速注入指定区域(图9c)，完毕后5秒内注入5ml生理盐水冲洗工作通道。撤去注胶系统，连接普通进水管。观察凝胶位置(图9d)，用
Figure PCTCN2022092370-appb-000010
或
Figure PCTCN2022092370-appb-000011
取石网篮取出胶体(图9e)。检查肾盂和各肾盏，取尽凝胶。
手术效果：术中取出粘附大量结石碎块的凝胶组合物(图9f)，而这些结石碎块在未使用该凝胶组合物的情况下，无法在术中取出体外，需患者术后自行排出。术后1日复查中下腹CT，未见明显结石残留(图8b-d)。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术 方案的范围。

Claims (10)

  1. 一种用于提高泌尿系统结石碎石取石手术效率、协助清除肾脏收集系统内结石碎块的凝胶组合物，其特征在于，包括A组分和B组分；
    A组分包含：纤维蛋白原和纤维蛋白稳定因子；
    B组分包含：凝血酶；
    其中，A组分和/或B组分中添加有Ca 2+。
  2. 根据权利要求1所述的凝胶组合物，其特征在于，A组分和/或B组分中添加有血纤维蛋白溶酶原；优选血纤维蛋白溶酶原的浓度为浓度D的1/256～1，优选为浓度D的1/64～1，优选为浓度D的1/45～1/20或1/32～1/8，优选为浓度D的1/35～1/25或1/10～1/8，优选为浓度D的1/32或1/10；所述浓度D为0.1mg/ml-1mg/ml。
  3. 根据权利要求1所述的凝胶组合物，其特征在于，在A组分和/或B组分中添加有无细胞毒性的染色剂；优选在B组分中添加有无细胞毒性的染色剂。
  4. 根据权利要求3所述的凝胶组合物，其特征在于，所述染色剂包括亚甲蓝、叶绿素或吲哚青绿；优选所述染色剂为亚甲蓝；进一步优选亚甲蓝的浓度为20.0μg/ml-60.0μg/ml。
  5. 根据权利要求1所述的凝胶组合物，其特征在于，所述纤维蛋白原和纤维蛋白稳定因子来源于人或动物血液或血液制品。
  6. 根据权利要求1所述的凝胶组合物，其特征在于，A组分中，还包含生理盐水；纤维蛋白原和纤维蛋白稳定因子被稀释在生理盐水中。
  7. 根据权利要求2所述的凝胶组合物，其特征在于，所述凝血酶和血纤维蛋白溶酶原来源于人或动物血液或血液制品。
  8. 根据权利要求1所述的凝胶组合物，其特征在于，B组分中，还包含生理盐水；凝血酶被稀释在生理盐水中。
  9. 根据权利要求1-8中任一项所述的凝胶组合物，其特征在于：
    A组分中纤维蛋白原和纤维蛋白稳定因子的总浓度为浓度A的1/256～1，优选为浓度A的1/64～1，优选为浓度A的1/45～1/20或1/32～1/8， 优选为浓度A的1/35～1/25或1/10～1/8，优选为浓度A的1/32或1/10；所述浓度A为1.8-3.8mg/ml，优选浓度A为3.8mg/ml；
    和/或，B组分中凝血酶的浓度为浓度B的1/256～1，优选为浓度B的1/64～1，优选为浓度B的1/45～1/20或1/32～1/8，优选为浓度B的1/35～1/25或1/10～1/8，优选为浓度B的1/32或1/10；所述浓度B为15.0-21.7IU/ml，优选浓度B为20IU/ml；
    和/或，B组分中凝血酶的用量与A组分中纤维蛋白原和纤维蛋白稳定因子的总用量之比为(15.0-21.7)IU:(1.8-3.8)mg；优选为20IU:3.8mg；
    和/或，A组分和/或B组分中，所述Ca 2+的浓度为浓度C的1/250～1，优选为浓度C的1/64～1，优选为浓度C的1/45～1/20或1/32～1/8，优选为浓度C的1/35～1/25或1/10～1/8，优选为浓度C的1/32或1/10；所述浓度C为5mM。
  10. 如权利要求1-9项中任一所述的凝胶组合物在制备用于清除经碎石取石术后残留的结石碎块的药物中的应用。
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