CN108273136B - A kind of hydration stomach takes off cell biological sticking patch and preparation method and application - Google Patents

A kind of hydration stomach takes off cell biological sticking patch and preparation method and application Download PDF

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CN108273136B
CN108273136B CN201810337490.2A CN201810337490A CN108273136B CN 108273136 B CN108273136 B CN 108273136B CN 201810337490 A CN201810337490 A CN 201810337490A CN 108273136 B CN108273136 B CN 108273136B
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sticking patch
stomach
hydration
preparation
cell
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薛万江
冯亮
郭益冰
毛勤生
胡懿林
冯盈
肖明兵
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Affiliated Hospital of Nantong University
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    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

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Abstract

The present invention relates to a kind of hydration stomaches to take off cell biological sticking patch and preparation method and application, the xenogenesis stomach acellular matrix sticking patch (Hydrated xenogeneic decellularized gastric matrix, HDGM) via the de- cell method preparation of hydration, it is dyed by HE, Masson dyeing, immunohistochemistry, DNA content and glycosaminoglycan content detection, CCK8 method, scanning electron microscope, the evaluation of biological nano dimple analysis, the result shows that main cell epimatrix composition is relatively stable, micro-structure retains intact, biomechanical property is stable and tissue compatible is without obvious cytotoxicity；Rat stomach local defect model is further constructed, interior evaluating HDGM has remarkable result for gastric perforation repairing.Novel stomach of the present invention takes off that cell biological sticking patch preparation method is simple, quality controllable, bio-compatible no cytotoxicity, non-immunogenicity, can be used as ideal gastric perforation repairing biomaterial.

Description

A kind of hydration stomach takes off cell biological sticking patch and preparation method and application
Technical field
It can be used at nearly gastric antrum pylorus or cardia perforating patient's compared with large perforation, stomach neoplasm advanced stage the present invention relates to a kind of The preparation method that the novel stomach of heterologous source takes off cell biological sticking patch is applied with it.
Background technique
Gastric perforation is the clinical most common acute abdomen, is more common in gastroduodenal ulcer, stomach late tumor ulceration.Gastric perforation When generation, gastric content, bile and gastric acid easily leak into abdominal cavity and cause intra-abdominal contamination, cause peritonitis, peritoneal abscess etc., when serious Toxic shock can occur, cause multiple organ failure, operative treatment is prefered method.For body of stomach compared with large perforation, merely Neoplasty is often hampered by Tissue Blood for the tension with edematous tissue；And for, compared with large perforation, being adopted at nearly gastric antrum pylorus or cardia Often cause pylorus or preventricular stenosis with SIMPLE CLOSURE, if using subtotal gastrectomy instead, reflux stomach easily occurs in postoperative patient The sequelae such as scorching, Gastroesophageal reflux disease and dumping syndrome, seriously affect patients ' life quality；Stomach late tumor is worn Hole, oedema easily bleeding at perforation, primary repair and resection w is postoperative easily to occur perforation leakage again.Tissue defect repairing is carried out with biomaterial Certain progress is achieved, have has for the material of puncture repair at present: polyglycolic acid biomembrane, Fibrin Glue.
Ideal gastric perforation repairing biology patching material should have good biomechanical property, histocompatbility, without bright Aobvious cytotoxicity.It is hydrated stomach and takes off what cytoskeleton inclusion inter-species was guarded, such as collagen, fibronectin, laminin Various structures and functional protein have good 3d space network structure, and ideal growth can be provided for regeneration and repairs sky Between.
Summary of the invention
It can be used for repairing defect of gastric wall the purpose of the present invention is to provide one kind, operate simple, quality controllable It is hydrated the preparation method and application that stomach takes off cell biological sticking patch.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of hydration stomach takes off cell biological sticking patch, it is characterized in that: 1) under the conditions of dry weight in HDGM DNA content less than 50 Ng/mg, and obvious nucleus residual is had no after HE dyeing, DAPI dyeing；2) HDGM retain a variety of main cell epimatrixs at Point: including Collagen I, Collagen, Fibronectin, Fibulin-1, GAGs etc.；3) through cobalt60After irradiation sterilization Biomethanics, ultra microstructure are without substantially changeing and without obvious cytotoxicity.Cobalt60Irradiation sterilization.Carry out HE dyeing, Masson dye Color, immunohistochemistry, DNA content and glycosaminoglycan content detection, CCK8 method, transmission electron microscope, biological nano dimple analysis are commented Valence and the evaluation of internal repair efficiency.
A kind of hydration stomach takes off the preparation method of cell biological sticking patch, it is characterized in that: just by the healthy adult rat put to death Middle notch opens abdominal cavity, appears and successively ligatures the total arteriovenous of ilium, Bilateral Renal arteriovenous；Open thoracic cavity, descending aorta remaining needle It punctures, ligation superior vena cava, aorta ascendens bow；Through descending aorta arterial bolus injection heparin, PBS buffer is perfused；It rapidly will - 80 DEG C of rat for setting pipe are refrigerated to de- cell stage, thaw under the conditions of 4 DEG C, are eluted using eluant, eluent mixed solution；ddH2O is 4 Continue 24 h of irrigation under the conditions of DEG C；1.5 ml/min of flow velocity separates gastric tissue, obtains stomach biological sticking patch.
Injecting heparin content through descending aorta is 20-100U, most preferably 30 U.
The perfusion PBS buffer time is 20 min -2 h, and Best Times are about 0.5 h.
Eluant, eluent mixed solution is Triton X-100, lauryl sodium sulfate, ammonium hydroxide, DNA enzymatic, RNA enzyme, deoxycholic acid Two or more in sodium.
Eluant, eluent mixed solution is that 1% Triton X-100/0.1% ammonium hydroxide and DNA enzymatic combine；Infusion time is 6-24 H, best infusion time are 8 h;Perfusion flow velocity is 0.2-2 ml/min, and the best flow velocity that is perfused is 1.5 ml/min.
A kind of application being hydrated the de- cell biological sticking patch of stomach in preparation gastric perforation patching material.
The gastric perforation is perforation or the perforation of stomach neoplasm advanced stage at nearly gastric antrum pylorus or cardia.
The present invention takes off cytoskeleton sticking patch (HDGM) as new bio patching material using aqua oxidation stomach, can lack to stomach wall Damage is repaired.The biological sticking patch is prepared by carrying out the de- cell of aqua oxidation to gastric wall in rats, biological group of sticking patch It is conservative at ingredient and space structure, and the biomethanics of sticking patch is enough to bear physical stress inside and outside gastral cavity.To hydration oxygen Change sticking patch (HDGM) and carry out Co 60 sterilization treatment, space structure and biomethanics are not destroyed；It is carried out using HDGM sticking patch big Mouse gastric perforation model is repaired, and rat has no rejection, and postoperative gastrointestinal image inspection has no that contrast agent leaks；3 Histological characterization is carried out to mend HDGM sticking patch after week, there are a large amount of mucomembranous epithelial cells to cover at sticking patch, muscle fibre is gradually repaired It is multiple.HDGM sticking patch is expected to become a kind of ideal biomaterial for clinical treatment.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is that rat stomach takes off cell biological sticking patch tissue morphology schematic diagram；
H&E, which is shown, has no obvious cell residue；The dye display of tri- color of Masson has no that muscle fibre remains；Immunohistochemistry is shown Collagen I 、Collagen , Fibronectin, Fibulin-1 preferably retain.
Fig. 2 is DNA and GAG content analysis schematic diagram；
Under the conditions of dry weight HDGM and not de- groups of cells (Native) be respectively 39 ± 4.02 ng/mg and 3689 ± 422.9 ng/mg;GAG is respectively 89 ± 5.2 ng/mg and 127 ± 7.9 ng/mg.
Fig. 3 is ultra microstructure, biomethanics and Cytotoxic evaluation schematic diagram；Obvious destruction is had no after de- cell processing； Biomechanical is respectively 1962 ± 261 kPa, 856 ± 47 kPa before and after taking off cell；Biological sticking patch leaching liquor is without obvious Cytotoxicity.
Fig. 4 is histocompatbility evaluation schematic diagram；
Postoperative 3,7,14,21 d respectively by bracket sample take out it is fixed after row HE dye, after transplanting 3-7 days as the result is shown, The inflammatory cell of graft area starts to infiltrate, and 7-14 days or so arrival peaks, inflammation gradually subsides after 14-21 days.
Fig. 5 is internal repairing evaluation schematic diagram；
Postoperative 24 h upper digestive tract radiography (UGI) shows contrast-agent-free extravasation, and stomach form is good；Contrast agent is suitable after 30 min Benefit enters small intestine, and alimentary canal is unobstructed；Postoperative 3 d, 7 d, 14 d, 21 d obtain the defect repairing surface of a wound after implementing air embolism execution Sample, row HE dyeing observation.
Specific embodiment
The present invention is further explained in the light of specific embodiments.
Animal used in this experimental example is SD rat, prepares the method for hydration biological sticking patch using the present embodiment and repairing is evaluated It is as follows:
(1) animal Preoperative Method
Healthy adult SD rat, 250-300 g, male and female are unlimited, and preoperative third day is reduced diet, give within preoperative second day Glucose solution is repeatedly fed on a small quantity, preoperative fasting in 1 day, and preoperative 4 h prohibits water, postoperative 24 h fasting.Surgical instrument conventional high-pressure Steam sterilizing, preoperative experimental site air purifying and sterilizing；Surgical staff operation abide by asepsis principle, operation with Standard surgical without Bacterium mode carries out.With 10% chloraldurate with 1 ml/100 g row intraperitoneal injection of anesthesia, povidone-iodine disinfection spreads aseptic towel.
(2) hydration biological sticking patch preparation
SD rat median incision opens abdominal cavity, appears and successively ligatures the total arteriovenous of ilium, Bilateral Renal arteriovenous；Open chest Chamber, descending aorta retained needle puncture, ligation superior vena cava, aorta ascendens bow.Through descending aorta arterial bolus injection heparin 30 U, PBS Buffer 0.5 h of perfusion.It is refrigerated to de- cell stage by -80 DEG C of rat that have set pipe rapidly, is thawed under the conditions of 4 DEG C, 1% 8 h are perfused through descending aorta in Triton X-100/0.1% ammonium hydroxide, 2 h are perfused in DNA enzymatic；ddH2O continues that 24 h are perfused in 4 DEG C, 1.5 ml/min of flow velocity, separation gastric tissue are spare.
(3) Histological evaluation
HE dyeing: prepared test serum fixes 1 h, Gradient elution using ethanol, specimens paraffin embedding slices (5 through 4% paraformaldehyde μm), in 70 DEG C of baking 1h.After dewaxing, successively haematoxylin dyeing, 1% hydrochloride alcohol break up, eosin stains；Immunohistochemistry: stone After wax slice dewaxing dehydration, 0.05% pancreatin is added dropwise, 3%H is added dropwise in 37 DEG C of constant-temperature incubations2O2Solution is incubated for block endogenous peroxide Compound enzyme, 10% sheep blood serum close endogenous antigen.Primary antibody rabbit-anti rat Collagen is added dropwise、CollagenⅣ、 Fibronectin, Fibulin-1, CK, Actin, α-smooth, CD31, NF-200(1:100, Santa Cruz Biotechnology, USA), 4 DEG C of overnight incubations are added secondary antibody and are incubated at room temperature 2 h, the colour developing of DAB liquid；Masson trichrome stain: After paraffin section de-waxing dehydration, through haematoxylin dyeing, the differentiation of 1% hydrochloride alcohol, then dyed with Ponceaux acid fuchsin liquid, 1% phosphorus After the processing of molybdic acid aqueous solution, brilliant green dye liquor is redyed.All slices use neutral gum mounting, and Olympus sediments microscope inspection is taken the photograph Piece.
(4) DNA and GAGs content detection is evaluated
Normal gastric mucosa, hydration stomach take off each 30 mg of cell tissue sample, according to DNA content extracts kit The operation of (TIANGEN company) interpellation；It takes 10 mg to shred to mill, is added 56 DEG C of 1 ml of Proteinase K overnight, 90 DEG C of standings 10 min are handled according to GAGs kit (ASSAY company) interpellation.Stomach biological sticking patch described in patent, under the conditions of dry weight DNA content is 39 ± 4.02 ng/mg, and GAGs content is 89 ± 5.2 ng/mg.
(5) Ultrastructural observation is evaluated
Take about 5 × 5 × 5 mm of sample3It is fixed overnight to be placed in 2.5 ﹪ glutaraldehydes, PBS buffer rinses, and 1% osmic acid is protected from light Processing, gradient alcohol dehydration, embedding, slice, Electronic Speculum (JEM-1230, JEOL) observe tissue hyper-microstructure.
(6) bracket Cytotoxic evaluation
It is required to use Cell counting Kit 8(CCK-8, Beyotime Institute of according to specification Biotechnology bracket leaching liquor toxicity) is evaluated.0.1 g/ml sample is added DMEM culture medium and prepares bracket leaching liquor, stomach Epithelial cell (GES-1) is resuspended and is inoculated on 96 orifice plates, and fresh DMEM is added in control group, and 100 μ of leaching liquor is added in experimental group L detects 450 nm OD values respectively at 0,24 h, 48 h, 72 h, 96 h.
(7) preparation of gastric perforation model and in vivo repairing evaluation
Preoperative Method and anesthesia are the same, and iodophor disinfection upper abdomen skin is about 5 cm using median abdominal incision, before selecting antrum Wall forms the defective region of the cm of 1 cm × 1 without blood vessel fan section, cut-out stomach wall.It is continuously stitched using the non-absorbable thread of 4-0 It closes to seal sterile sticking patch and mends defective region, the non-absorbable thread of 3-0 closes abdominal cavity.The postoperative same day is placed in the receptacle of heater unit, It is kept for 26.0 DEG C -30.0 DEG C of room temperature, avoids postoperative hypothermia.All animals give 5% glucose 10 in postoperative subcutaneous injection Ml, physiological saline 10 ml, postoperative 8 h prohibit water, 48 h fasting.The postoperative 24 h row iodized oil alimentary canal of rat stomach puncture repair is made Shadow, chloral hydrate anesthesia, respectively through 0.5 ml iodized oil of Injection by stomach duct, in X after 5 min, 10 min, 15 min, 30 min Line photographic apparatus (DR) has an X-rayed read tablet.
It should be pointed out that preparation method described in the present embodiment, it may also be used for prepare new zealand white rabbit, beasle dog, small The stomach of other animals such as type pig takes off cell biological sticking patch.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills Art field, is included within the scope of the present invention.

Claims (6)

1. a kind of hydration stomach takes off cell biological sticking patch, it is characterized in that: 1) under the conditions of dry weight in HDGM DNA content less than 50 Ng/mg, and obvious nucleus residual is had no after HE dyeing, DAPI dyeing；2) HDGM retain a variety of main cell epimatrixs at Point: including Collagen I, Collagen,Fibronectin,Fibulin-1,GAGs；3) through cobalt60It is raw after irradiation sterilization Material resources, ultra microstructure are without substantially changeing and without obvious cytotoxicity.
2. a kind of hydration stomach takes off the preparation method of cell biological sticking patch, it is characterized in that: the healthy adult rat put to death is hit exactly Notch opens abdominal cavity, appears and successively ligatures the total arteriovenous of ilium, Bilateral Renal arteriovenous；Thoracic cavity is opened, descending aorta remaining needle is worn Thorn, ligation superior vena cava, aorta ascendens bow；Through descending aorta arterial bolus injection heparin, PBS buffer is perfused；It will set rapidly - 80 DEG C of rat of pipe are refrigerated to de- cell stage, thaw under the conditions of 4 DEG C, are eluted using eluant, eluent mixed solution；ddH2O is at 4 DEG C Under the conditions of continue 24 h of irrigation；1.5 ml/min of flow velocity separates gastric tissue, obtains stomach biological sticking patch.
3. hydration stomach according to claim 2 takes off the preparation method of cell biological sticking patch, it is characterized in that: pushing away through descending aorta Note heparin content is 20-100U.
4. hydration stomach according to claim 2 takes off the preparation method of cell biological sticking patch, it is characterized in that: perfusion PBS is slow The fliud flushing time is 20 min -2 h.
5. hydration stomach according to claim 2 takes off the preparation method of cell biological sticking patch, it is characterized in that: eluant, eluent mixing is molten Liquid is Triton X-100, lauryl sodium sulfate, ammonium hydroxide, DNA enzymatic, RNA enzyme, two kinds or two kinds in NaTDC with On.
6. hydration stomach according to claim 5 takes off the preparation method of cell biological sticking patch, it is characterized in that: eluant, eluent mixing is molten Liquid is that 1% Triton X-100/0.1% ammonium hydroxide and DNA enzymatic combine；Infusion time is 6-24 h;It is 0.2-2 that flow velocity, which is perfused, ml/min。
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