CN107119008A - A kind of young porcine islet isolation method and separation digestive juice - Google Patents

A kind of young porcine islet isolation method and separation digestive juice Download PDF

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CN107119008A
CN107119008A CN201710313206.3A CN201710313206A CN107119008A CN 107119008 A CN107119008 A CN 107119008A CN 201710313206 A CN201710313206 A CN 201710313206A CN 107119008 A CN107119008 A CN 107119008A
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digestive juice
pancreas
tissue
islet
young
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CN107119008B (en
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王维
马小倩
王佳
李桑
徐畅
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Central South University
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Abstract

The invention discloses a kind of young porcine islet isolation method and separation digestive juice.Described digestive juice with the addition of DNase I, neutral proteinase, I, III Collagenase Type, CaCl on the basis of use with the addition of conventional digestion enzyme as liquid phase using basal medium or balanced salt solution2, Trolox, growth hormone release inhibiting hormone etc..Change traditional perfusion digestion method when making isolated pancreatic islet simultaneously, be changed into shredding tissue digestion method, to improve young pig pancreas islet yield.The present invention can stablize in the case where not influenceing porcine islet function and improve islet cells yield, reduction financial cost, and easily operated, reduce contamination probability.

Description

A kind of young porcine islet isolation method and separation digestive juice
Technical field
The invention belongs to the technical field of porcine islet separation, a kind of young porcine islet isolation new method is specifically related to And separation digestive juice.
Background technology
Patients with type Ⅰ DM is a kind of metabolic disease of global serious threat human health, immune mediating with selectivity Destroy with the characteristics of beta Cell of islet, patient needs exogenous insulin life-long therapy.But various complication cause minimal invasive treatment, Financial burden exacerbation and the rising of the death rate.From after the success of Edmonton schemes, pancreatic islets transplantation receives attracting attention for the world, total The method and immunosuppressant scheme that transplanting is improved on the basis of global pancreatic islets transplantation achievement in research have been tied, has been IDDM disease People brings the hope of healing.Human pancreatic's pancreas islet of allograft has proved to be a kind of feasible selection and treats I type The method of diabetes, can improve control patient blood glucose, reduce insulin requirements, reduce the generation of hypoglycemia after transplanting.Pancreas islet Transplanting has become the effective ways of IDDM patient at present, but people's pancreas islet source is difficult, and isolated pancreatic islet technology is also endless It is kind, and there is the risk of transmission.Recently, pig pancreatic islets transplantation is proved to be a feasibility for solving the shortage of pancreas islet source Scheme.
The factor of selection Human islet transplanting or Xenografts for Treatment IDDM has many, including security, clinic Pick-up rate of curative effect and pancreas islet etc..Islets Xenotransplantation is that insulin-dependent diabetes mellitus is treated in application pancreatic islets transplantation in recent years A study hotspot, homograft and stem cell transplantation have its more limitation, and pork insulin and actrapid monotard's difference One non-conservative region amino acid sequence, can bring into normal play function in human body, and originates and relatively enrich, thus be considered as current Suitable donor source.Though young pig pancreas incomplete development is ripe, compared with neonatal pig, its pancreas volume, weight etc. are all Larger, pancreatic islet alpha-Gal antigen presentations are relatively weak, and pancreas islet maturity is of a relatively high, therefore it is thin to obtain a large amount of ripe pancreas islet Born of the same parents.But, the pancreas islet of young pig lacks complete coating and surround, and separation and purge process are complex, Pancreas Islet Structure and function Integrality is easily destroyed in the process, so as to influence pancreas islet yield and function.How efficiently, successfully separation pig pancreas islet is thin Born of the same parents just turn into the restraining factors of pig pancreatic islets transplantation, and traditional separation method has that pancreas islet yield, purity is low, operability is weak or warp The problems such as cost of helping is high, so as to cause the waste of each side resource.Therefore it further need to study and improve young porcine islet isolation Purification technique, is moved with obtaining the good islet cells of enough structural integrities, function so as to meet big animal implant tests textured and clinic The need for plant.This research method is intended to inquire into and improves young porcine islet isolation purification technique.By shred tissue digestion method with And the digestive juice being especially formulated is used in combination, to optimize young pig pancreas islet preparation method, pancreas islet yield is improved.
The content of the invention
It is an object of the invention to provide one kind in the case where not influenceing islet function, it can stablize and improve pancreas islet yield Young porcine islet isolation method and the digestive juice for separation, this method are easily operated, reduce contamination probability.
A kind of young porcine islet isolation method, is digested by the way of tissue is shredded, and digestive juice is with basis training It is to add DNase I, neutral proteinase, I, III type on the basis of liquid phase with the addition of conventional digestion enzyme to support base or balanced salt solution Clostridiopetidase A, CaCl2, Trolox, growth hormone release inhibiting hormone.
5~10U/ml DNase I, 12000-4000u/g neutral proteinases, concentration are added in described digestive juice is 0.1~0.5mg/ml I, III Collagenase Type, 1~10mM CaCl2, 1~10mM Trolox, 1~5ug/ml growth hormone release inhibiting hormones；Institute The conventional digestion enzyme stated is V Collagenase Type, and addition concentration is 0.1~1mg/ml；Described basal medium is RPMI1640, Described balanced salt solution is HBSS.
As a further improvement, the concentration of I, III Collagenase Type is 0.5mg/ml, CaCl in described digestive juice2For 1mM；Trolox is 5mM, and growth hormone release inhibiting hormone is 2.5ug/ml.
As a further improvement, pancreatic tissue is shredded to organizing a diameter of 1~5mm, plus digestive juice processing.
As a further improvement, digestive juice and liquid-solid ratio 20~50ml/g pancreas of pancreatic tissue.
As a further improvement, pancreas is first shredded roughly, handled with HBSS buffer solutions, be further continued for using digestive juice after shredding Processing.
As a further improvement, plus 10ml buffer solutions, scissors shreds a diameter of 3~5mm of pancreatic tissue, takes pancreas weight After 2~5g, plus buffer solution, to 50ml, mixing, 200rpm centrifugation 1min abandon 30~35ml of supernatant, are repeated once；Shred in tissue Plus 25~30ml digestive juices after continue to cut to organizing a diameter of 1~5mm；By the tissue shredded plus digestive juice to 100ml.
As a further improvement, following steps are specifically included：
1) cold ischemia time ＜ 30min young pig pancreas is got out；
2) pancreas is successively oozed into the tincture of iodine, be transferred to after physiological saline in sterile tray, pallet keeps ice bath；
3) lymph and connective tissue of pancreas are removed；
4) pancreas is transferred in 50ml sterile centrifugation tubes and weighs and record；
5) 10ml buffer solutions are added, scissors shreds a diameter of 3~5mm of pancreatic tissue, takes pancreas 2~5g of weight, plus buffer solution To 50ml, mix, 200g centrifugation 1min abandon 30~35ml of supernatant, are repeated once；
6) shred in tissue Jia 25~30ml digestive juices after continue to cut to organizing a diameter of 1~5mm；
7) tissue shredded is transferred in 250ml conical flasks, plus digestive juice is to 100ml；
8) taper bottle closure, 37 DEG C, 100rpm 15~20min of concussion, digestive juice adds neutralizer to 250ml termination when becoming muddy Digestion；Described neutralizer is the HBSS buffer solutions containing 5% Swine serum；
9) 40 mesh metal screens are placed on beaker, filtration step 8) the tissue digestion liquid for preparing, and use 10ml
Syringe piston is ground on metal screen, until having filtered all digestive juices；
10) neutralizer is cleaned 2 times, 200rpm, 2min centrifugation, abandons supernatant；
11) cold HBSS is resuspended, 200rpm, 2min centrifugation, abandons supernatant, is repeated 2 times；
12) precipitate, 200rpm, 2min centrifugation is resuspended in the maturation medium for adding precooling；Described maturation medium For the RPMI1640 culture mediums containing 10% Swine serum.
13) histocyte is resuspended in maturation medium, and average mark is into blake bottle, 37 DEG C, 5%CO2Culture, every pig point 4 ~6 T-175 suspension blake bottle.
The innovative point of the present invention is as follows：
DNAse acts on extracellular dna, and extracellular dna plays an important roll to the biofilm structure of most species, to biomembrane Being formed has facilitation, and the starting of biomembrane can be influenceed to stick maturation with biomembrane.The mature biology of DNAse decomposable asymmetric choice nets Film, reduces the adhesion of biomembrane.Destruction to DNA causes the reduction of intracellular matrix/extracellular polymeric to play a role, and prevents Released dna causes solution excessively viscous after only digesting.Conventional digestion enzyme makes many hydrolase polypeptides of the proline of cytoplasm so that cell from Dissipate.
Neutral proteinase, can Hydrolysis of tissue albumen, be the albumen that a class acts on protein peptide bond in neutral conditions Protein, can be hydrolyzed into polypeptide or free amino acid, contribute to the discrete of cell by enzyme, while it is free can also effectively to remove hydroxyl Base, weakens the response to oxidative stress produced during proteolysis；Ith, III Collagenase Type acts on collagen Gly-Xaa-Yaa The connection of lysine in (- Gly-) repetitive sequence/between arginine and glycine, from C-terminal hydrolytic collagen material, coordinates V type glue The characteristic of protoenzyme hydrolytic collagen since N-terminal, can effectively improve the utilization rate of digestive ferment, accelerate cell separation process, improve and divide From efficiency；Ca2+It can be combined with collagenase P KD with CBD areas calcium binding sites, play stable molecule conformation, improve clostridiopetidase A The purpose of stability, can effectively improve digestive ferment utilization rate；Trolox, can prevent oxidation caused by peroxynitrite Stress, there is anti-apoptotic, reduces the effect of oxidative stress, can effectively prevent from producing oxyradical in digestion process to separation Islet cells produce injury.The exocrine function of pancreas can effectively be suppressed by adding growth hormone release inhibiting hormone, reduce tryptic activity, be prevented Only exocrine gland produces the characteristics of damaging action present invention combines various enzymes to islet cells, and anti-oxidation stress is used in combination, resists Apoptosis, suppression exocrine gland medicine etc., are used in combination during islet cells is separated, improve pancreas islet yield to reach, carry High digestive ferment utilization rate, reduction islet cells separates the purpose of financial cost.
Except the digestive juice for the new formula that the present invention is used, digested while coordinating by the way of tissue is shredded, can It is 1.67 × 10 to obtain yield4IEQ/g, purity are 75.3% pancreas islet, have conspicuousness relative to traditional separation pancreas islet method Raising.Islet function testing result shows that its SI of islet cells separated using the present invention is 2.27, and islet cell function is good Good, there was no significant difference for the islet cell function obtained with conventional separation methods, and operating process is simple, easy.
Compared with the prior art, the advantage that the present invention is obtained：
1st, Unit Weight pancreatic islet pick-up rate conspicuousness is improved；
2nd, the cell bunchiness phenomenon produced by under common digestion and situation about excessively digesting are successfully avoid, so as to reduce Islet cells loses；
3rd, the pancreas islet of separation is of moderate size, the need for meeting big animal implant tests textured and clinical transplantation；
4th, compared to conventional perfusion digestion method, it is easy to operate；
5th, the utilization rate of digestive juice, the young porcine islet separation financial cost of reduction can be effectively improved using the present invention.
Brief description of the drawings
Fig. 1 is various concentrations I, III Collagenase Type coordinates V Collagenase Type to obtain islet cells under different digestions time Efficiency and quality (100 ×)；
As a result, it was confirmed that in the case where concentration is 0.5mg/ml, separative efficiency is grouped better than other, therefore subsequent experimental is equal in Fig. 1 Concentration is used to coordinate V Collagenase Type pancreas digested for 0.5mg/ml I, III Collagenase Type.Digestion time is optimal with 17 minutes.
Fig. 2 is that AO/EB detects that various concentrations I, III Collagenase Type coordinate V Collagenase Type under identical digestion time, cell Apoptosis situation (100 ×)；
As a result, it was confirmed that relative to other two groups in Fig. 2, under concentration is 0.1mg/ml and 0.5mg/ml, Cell viability preferably, The green fluorescence for representing living cells is more, and the red fluorescence for representing dead cell is less, but obtains pancreas islet under 0.1mg/ml concentration Digestion time is longer needed for cell, adds the time of hot ischemic, is unfavorable for later stage culture transplanting and uses.
Fig. 3 is DTZ dyeing islet cells forms (100 ×) after separation 48h；
In Fig. 3, experimental group is the isolated islet cells of the inventive method, and control group is poor compared with the method for the present invention It is not to be used only V Collagenase Type as the isolated islet cells of digestive juice.
Fig. 4 is that various concentrations Trolox is used cooperatively down with growth hormone release inhibiting hormone, Intra-islet Apoptosis situation；
Fig. 4 results are shown：Under conditions of Trolox and growth hormone release inhibiting hormone is added, the green fluorescence for representing living cells is more, The red fluorescence for representing dead cell is less, and preferably, from the aspect of financial cost, selection 5mM Trolox coordinate Cell viability 2.5ug/ml growth hormone release inhibiting hormones are used.
Fig. 5 is islet cells yield；
In Fig. 5, experimental group is the isolated islet cells of the inventive method, and control group 1 is compared with the method for the present invention Difference is to be used only V Collagenase Type as the isolated islet cells of digestive juice, and control group 2 is to be disappeared using tradition perfusion Change method coordinates V Collagenase Type to be used as the isolated islet cells of the method for digestive juice.
Fig. 6 is that islet cells size compares (um).
In Fig. 6, experimental group is the isolated islet cells of the inventive method, and control group is poor compared with the method for the present invention It is not to be used only V Collagenase Type as the isolated islet cells of digestive juice.The islet cells obtained using the present invention is existed It is more suitable for later stage transplanting needs in size.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1：The separation of young pig pancreas islet：
1st, the containers such as the centrifuge tube, conical flask, pallet to be used are got out, and carry out mark；
2nd, young pig pancreas (cold ischemia time ＜ 30min) is separated；
3rd, pancreas is successively oozed into the tincture of iodine, be transferred to after physiological saline in sterile tray, pallet keeps ice bath；
4th, pancreas unnecessary lymph and connective tissue are removed；
5th, pancreas is transferred in 50ml sterile centrifugation tubes and weighs and record；
6th, 10ml buffer solutions are added, scissors is cut after pancreas 1min, plus buffer solution, to 50ml, mixing, 200g centrifugation 1min are abandoned Clear 30~35ml, is repeated once；
7th, shred in tissue Jia 25~30ml digestive juices after continue to cut 10min to organizing a diameter of 1~5mm；
8th, the tissue shredded is transferred in 250ml conical flasks, plus digestive juice is to 100ml；
9th, taper bottle closure, 37 DEG C, 100rpm 15~20min of concussion, digestive juice adds neutralizer to 250ml termination when becoming muddy Digestion；
10th, metal screen is placed on beaker, 4~5ml/ times filtering, and with 10ml syringe pistons on metal screen It is ground, until having filtered all digestive juices (if pancreatic tissue is more on filter screen should change filter screen in time)；
11st, neutralizer cleaning filter screen to cumulative volume reaches 500ml (250ml+250ml), and supernatant is abandoned in 200g, 2min centrifugation；
12nd, cold HBSS is resuspended and merges precipitate, 200g, 2min centrifugation, abandons supernatant, is repeated 2 times；
13rd, precipitate, 200g, 2min centrifugation is resuspended in the maturation medium for adding precooling；
14th, histocyte is resuspended in maturation medium, and average mark is into blake bottle, 37 DEG C, 5%CO2(every pig is general for culture 4~6 T-175 suspension blake bottle can be divided).
Embodiment 2：Pancreas islet form, quantity detection
Material and reagent：50ml centrifuge tubes, 250ml centrifuge tubes, 250ml conical flasks, 500um metal screens, 10ml injections Device, metal tray, water-bath, incubator, centrifuge tube shelf, scissors, haemostatic clamp, blake bottle, beaker, JPI complete mediums, pig blood Clearly, HBSS, antibiotic, the tincture of iodine, DNA enzymatic, physiological saline etc.
AO/EB detects Intra-islet Apoptosis：Acridine orange (AO), ethidium bromide (EB) each lmg are measured, 10ml pH are dissolved in respectively Be allowed to be made into 100 μ g/ml mother liquor in 7.2PBS, 4 DEG C be kept in dark place, it is standby.Use preceding mixed in equal amounts.The μ l of cell suspension 100 are taken, Add the μ l of AO/EB dyestuffs 4 to mix, take one after another drop of in slide, fluorescence microscopy Microscopic observation result.As a result Fig. 1,2,4 are seen.
Form, number and the purity of DTZ dyeing observation islet cells
10mg DTZ are dissolved in 10ml dimethyl sulfoxide (DMSO) (DMSO), degerming rear point with 0.22 μm of aperture membrane filtration Dress is stored in -20 DEG C of refrigerators.During normal dyeing, mixed per 1ml islet cells suspension with 10 μ l DTZ storing liquids, in 37 DEG C Microscopy after being incubated 10 minutes, islet cells is dyed to observe the form of cell after scarlet, as a result sees Fig. 3.
Islet cells group number/cell mass sum × 100% of pancreas islet purity=DTZ stained positives.The present invention obtains yield For 1.67 × 104IEQ/g, purity are 75.3% pancreas islet, are had significantly relative to traditional separation pancreas islet method (i.e. control group 2) The raising of property, is as a result shown in Fig. 3.
Embodiment 3：Islet function is detected
1st, insulin release test detects the function of islet cells
When islet cells is cultivated to the 6th day and the 10th day respectively, 6000IEQ cell suspension natural sedimentation 6 is extracted respectively Cell is suctioned out after minute, is washed twice with the nutrient solution of low sugar containing 2.5mM and incubating cells is in 37 DEG C, 5%CO2Incubator 1 hour, Cell suspension is taken out by its natural sedimentation 6 minutes, every group of islet cells is divided into 2 parts, and every part is separated into 3 parallel holes, point Jia Ru not the sugared nutrient solution (Hyclone culture mediums) of 2ml low sugar containing 2.5mM nutrient solution (Hyclone culture mediums) and the 2ml height containing 25mM In in 6 hole culture dishes, 37 DEG C, 5%CO are put into2It is further cultured in incubator after 2 hours, centrifuging and taking supernatant passes through chemiluminescence Method detects insulin content.Calculate glucose stimulus index (SI).SI>1.8 represent islet cells function it is good.
Insulin release during glucose stimulus index (Stimulation index, SI)=25mM glucose cultures/ Insulin release during 2.5mM glucose cultures
The each group islet cells glucose stimulus index of table 1
Experimental group is the islet cells that the inventive method is prepared, and control group is for difference compared with the method for the present invention Using only V Collagenase Type as the isolated islet cells of digestive juice, as a result illustrate that two groups of result of the tests are more or less the same, enter One step illustrates that the inventive method does not influence porcine islet function.
2nd, Insulin/DNA detects insulin content
1200IEQ islet cells is extracted, every group of cell is divided into 2 parts after being suspended with 600 μ l PBS, and every part is separated into 2 Individual parallel hole is transferred in 2ml centrifuge tube respectively, wherein 1 part is separately added into 1ml Azol per hole and is detected for insulin, separately Outer 1 part does not add Azol cell suspension to be detected for DNA.
The configuration of 2.1 reagents
1) Azol configuration
RIA-BSA1.25g and acetic acid 57ml are dissolved in wiring solution-forming after being mixed in 500ml distilled water, 4 DEG C of refrigerators are put in Inside save backup.
2) FSA configuration
By NaCl 4.383g, NaH2PO4·H2O 0.780g、NaH2PO4·2H2O 0.881g and RIA-BSA0.25g is molten In being mixed in 500ml distilled water, wiring solution-forming after the pH value of solution is adjusted into 7.35, is put in 4 DEG C of refrigerators and saved backup.
2.2 insulin are detected
Above-mentioned addition 1ml Azol holes cell suspension is placed on ice, after being crushed 20 seconds with Ultrasonic Cell Disruptor, 50 μ are taken L supernatants add 500 μ l FSA in being put in drying in baking box in 2ml centrifuge tube after drying, be put in preservation in -20 DEG C of refrigerators, most Unify to detect insulin content by chemoluminescence method afterwards.
2.3DNA detection
The above-mentioned holes cell suspension unification for not adding Azol according to the operation of AxyPrep genomic DNA small volume of reagent boxes Illustrate to extract DNA, concrete operation step is as follows：
1) 250 μ l deionized water or PBS suspension cells is added.
2) 1min is stood at room temperature after adding 0.8 μ l RNase A, concussion 15s.
3) 150 μ l buffer C-L and 8 μ l proteinase K are added, concussion 1min is well mixed it, passed through Then centrifuge tube is put in 56 DEG C of water-bath 10min by centrifugation.
4) 350 μ l buffer P-D are added, 30s, 12,000 × g centrifugations are shaken using whirlpool to make it well mixed 10min。
5) prepared by DNA into pipe to be put among 2ml centrifuge tubes, the mixed liquor immigration in previous step is prepared among pipe, 12, 000 × g centrifuges 1min.
6) filtrate is discarded, is placed in pipe is prepared among original 2ml centrifuge tube, 500 μ l Buffer W1 are then added 12,000 × g centrifuges 1min.
7) filtrate is discarded, is placed in pipe is prepared among original 2ml centrifuge tube, 700 μ l Buffer W2 are then added 12,000 × g centrifuges 1min, uses identical method, and the Buffer W2 for adding 700 μ l washed once again.
8) filtrate is discarded, is placed in pipe is prepared among original 2ml centrifuge tube, 12,000 × g centrifugations 1min.
9) prepared by DNA into pipe to be put among another clean 1.5ml centrifuge tube, 100- is added in the center for preparing periosteum 200 μ l Eluent or deionized water, stands after 1min at room temperature, 12,000 × g centrifugation 1min eluted dnas.
10) DNase-free water dilutes 10 × DNA sample, and the quality and concentration of DNA sample utilize nucleic acid-protein analyzer To detect.
The comparison of the Insulin/DNA values of each group islet cells of table 2
Experimental group is the islet cells that the inventive method is prepared, and control group is for difference compared with the method for the present invention Using only V Collagenase Type as the isolated islet cells of digestive juice, as a result illustrate that two groups of result of the tests are more or less the same, enter One step illustrates that the inventive method does not influence porcine islet function.

Claims (10)

1. a kind of young porcine islet isolation method, it is characterised in that digested by the way of tissue is shredded, digestive juice be DNase I, neutral protein are added on the basis of with the addition of conventional digestion enzyme as liquid phase using basal medium or balanced salt solution Enzyme, I, III Collagenase Type, CaCl2, Trolox, growth hormone release inhibiting hormone.
2. young porcine islet isolation method according to claim 1, it is characterised in that add 5 in described digestive juice~ 10U/ml DNase I, 12000-4000u/g neutral proteinases, concentration be the I of 0.1~0.5mg/ml, III Collagenase Type, 1~ 10mM CaCl2, 1~10mM Trolox, 1~5ug/ml growth hormone release inhibiting hormones；Described conventional digestion enzyme is V Collagenase Type, addition Concentration is 0.1~1mg/ml；Described basal medium is RPMI1640, and described balanced salt solution is HBSS.
3. young porcine islet isolation method according to claim 1, it is characterised in that I, III type glue in described digestive juice The concentration of protoenzyme is 0.5mg/ml, CaCl2For 1mM；Trolox is 5mM, and growth hormone release inhibiting hormone is 2.5ug/ml.
4. young porcine islet isolation method according to claim 1, it is characterised in that shred pancreatic tissue to organizing diameter Handled for 1~5mm, plus digestive juice.
5. young porcine islet isolation method according to claim 1, it is characterised in that digestive juice and the liquid of pancreatic tissue are solid Than 20~50ml/g pancreas.
6. young porcine islet isolation method according to claim 1, it is characterised in that first shred pancreas roughly, use HBSS Buffer solution processing, is further continued for after shredding being handled with digestive juice.
7. young porcine islet isolation method according to claim 1, it is characterised in that plus 10ml buffer solutions, scissors is shredded A diameter of 3~the 5mm of pancreatic tissue, is taken after 2~5g of pancreas weight, plus buffer solution, to 50ml, mixing, 200rpm centrifugation 1min are abandoned 30~35ml of supernatant, is repeated once；Shred in tissue Jia 25~30ml digestive juices after continue to cut to organizing a diameter of 1~5mm；Will The tissue plus digestive juice shredded is to 100ml.
8. young porcine islet isolation method according to claim 1, it is characterised in that specifically include following steps：
1) cold ischemia time ＜ 30min young pig pancreas is got out；
2) pancreas is successively oozed into the tincture of iodine, be transferred to after physiological saline in sterile tray, pallet keeps ice bath；
3) lymph and connective tissue of pancreas are removed；
4) pancreas is transferred in 50ml sterile centrifugation tubes and weighs and record；
5) 10ml buffer solutions are added, scissors shreds a diameter of 3~5mm of pancreatic tissue, takes pancreas 2~5g of weight, plus buffer solution is extremely 50ml, mixing, 200g centrifugation 1min, abandon 30~35ml of supernatant, are repeated once；
6) shred in tissue Jia 25~30ml digestive juices after continue to cut to organizing a diameter of 1~5mm；
7) tissue shredded is transferred in 250ml conical flasks, plus digestive juice is to 100ml；
8) taper bottle closure, 37 DEG C, 100rpm 15~20min of concussion, digestive juice adds neutralizer to stop to disappear to 250ml when becoming muddy Change；
Described neutralizer is the HBSS buffer solutions containing 5% Swine serum；
9) 40 mesh metal screens are placed on beaker, filtration step 8) the tissue digestion liquid for preparing, and use 10ml syringe pistons It is ground on metal screen, until having filtered all digestive juices；
10) neutralizer is cleaned 2 times, 200rpm, 2min centrifugation, abandons supernatant；
11) cold HBSS is resuspended, 200rpm, 2min centrifugation, abandons supernatant, is repeated 2 times；
12) precipitate, 200rpm, 2min centrifugation is resuspended in the maturation medium for adding precooling；Described maturation medium be containing There are the RPMI1640 culture mediums of 10% Swine serum.
13) histocyte is resuspended in maturation medium, and average mark is into blake bottle, 37 DEG C, 5%CO2Culture, every pig point 4~6 T-175 suspension blake bottle.
9. a kind of young porcine islet isolation digestive juice, it is characterised in that digestive juice is with basal medium or balance salt Solution is to add DNase I, neutral proteinase, I, III Collagenase Type, CaCl on the basis of liquid phase with the addition of conventional digestion enzyme2、 Trolox, growth hormone release inhibiting hormone.
10. young porcine islet isolation digestive juice according to claim 9, it is characterised in that add in described digestive juice It is the I of 0.1~0.5mg/ml, III Collagen Type VI to enter 5~10U/ml DNase I, 12000-4000u/g neutral proteinases, concentration Enzyme, 1~10mM CaCl2, 1~10mM Trolox, 1~5ug/ml growth hormone release inhibiting hormones；Described conventional digestion enzyme is V Collagen Type VI Enzyme, addition concentration is 0.1~1mg/ml；Described basal medium is RPMI1640, and described balanced salt solution is HBSS.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609439A (en) * 2018-12-25 2019-04-12 中国医学科学院北京协和医院 Single celled method is separated from non-normal tissue

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033460A (en) * 2007-01-19 2007-09-12 牛申 Method of separating, purifying and cultivating live pig pancreatic island cell
CN103361298A (en) * 2012-03-27 2013-10-23 中国科学院大连化学物理研究所 Separation and extraction fluid suitable for pig pancreas islet and preparation and application thereof
CN103429733A (en) * 2010-10-22 2013-12-04 细胞与组织系统股份有限公司 Cultured pancreas islets
CN103509750A (en) * 2012-06-29 2014-01-15 中国科学院大连化学物理研究所 Method for separating and purifying mammal insulin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033460A (en) * 2007-01-19 2007-09-12 牛申 Method of separating, purifying and cultivating live pig pancreatic island cell
CN103429733A (en) * 2010-10-22 2013-12-04 细胞与组织系统股份有限公司 Cultured pancreas islets
CN103361298A (en) * 2012-03-27 2013-10-23 中国科学院大连化学物理研究所 Separation and extraction fluid suitable for pig pancreas islet and preparation and application thereof
CN103509750A (en) * 2012-06-29 2014-01-15 中国科学院大连化学物理研究所 Method for separating and purifying mammal insulin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A.-Y. QIAO等: "Isolation and Purification of Islet Cells From Adult Pigs", 《TRANSPLANTATION PROCEEDINGS》 *
周文学: "单纯酶与复合酶对成猪胰岛分离效果的研究", 《中华实验外科杂志》 *
李建国 等: "胰岛移植的研究进展", 《中国伤残医学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609439A (en) * 2018-12-25 2019-04-12 中国医学科学院北京协和医院 Single celled method is separated from non-normal tissue
CN109609439B (en) * 2018-12-25 2020-11-06 中国医学科学院北京协和医院 Method for isolating single cells from abnormal tissue

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