CN107119008B - It is a kind of youth porcine islet isolation method and separation use digestive juice - Google Patents
It is a kind of youth porcine islet isolation method and separation use digestive juice Download PDFInfo
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Abstract
The invention discloses a kind of young porcine islet isolation method and separation digestive juices.The digestive juice is added to DNase I, neutral proteinase, I, III Collagenase Type, CaCl on the basis of use is added to 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, become shredding tissue digestion method, to improve young pig pancreas islet yield.The present invention can stablize in the case where not influencing porcine islet function and improve islet cells yield, reduce economic cost, and easily operated, reduce contamination probability.
Description
Technical field
The invention belongs to the technical fields of porcine islet separation, are specifically related to a kind of young porcine islet isolation new method
And digestive juice is used in separation.
Background technique
Patients with type Ⅰ DM is a kind of global metabolic disease for seriously threatening human health, immune mediating with selectivity
It destroys with the characteristics of beta Cell of islet, patient needs exogenous insulin life-long therapy.But various complication cause minimal invasive treatment,
The rising of financial burden exacerbation and the death rate.From after the success of Edmonton scheme, pancreatic islets transplantation receives attracting attention for the world, total
The method and immunosuppressant scheme for improving transplanting on the basis of global pancreatic islets transplantation research achievement have been tied, has been Type I diabetes disease
People brings the hope of healing.Human pancreatic's pancreas islet of allograft has proved to be a kind of I type of feasible selection treatment
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 Type I diabetes patient at present, but people's pancreas islet source is difficult, and isolated pancreatic islet technology is also endless
It is kind, and there are the risks of transmission.Recently, pig pancreatic islets transplantation is proved to be a feasibility for solving the shortage of pancreas islet source
Scheme.
There are many factors for selecting Human islet's transplanting or Xenografts for Treatment Type I diabetes, including safety, clinic
Curative effect and the pick-up rate of pancreas islet etc..Islets Xenotransplantation is to treat insulin-dependent diabetes mellitus using pancreatic islets transplantation in recent years
A research hotspot, the limitation that homograft and stem cell transplantation have its more, and pork insulin and actrapid monotard difference
One non-conservative region amino acid sequence can bring into normal play function in human body, and source is relatively abundanter, thus be considered current
Suitable donor source.Though young pig pancreas incomplete development is mature, compared with neonatal pig, pancreas volume, weight etc. are all
Larger, pancreatic islet alpha-Gal antigen presentation is relatively weak, and pancreas islet maturity is relatively high, therefore it is thin to can get a large amount of mature pancreas islet
Born of the same parents.But the pancreas islet of young pig lacks complete coating and surround, separation and purification process are complex, Pancreas Islet Structure and function
Integrality is easily destroyed in the process, to influence pancreas islet yield and function.How efficiently, successfully separation pig pancreas islet is thin
Born of the same parents just become the restraining factors of pig pancreatic islets transplantation, traditional separation method there are pancreas islet yield, purity is low, operability is weak or warp
It helps the problems such as at high cost, to cause the waste of various aspects resource.Therefore it further need to study and improve young porcine islet isolation
Purification technique meets big animal implant tests textured and clinical shifting to obtain the good islet cells of enough structural integrities, function
The needs of plant.This research method is intended to inquire into and improve young porcine islet isolation purification technique.By shred tissue digestion method with
And the digestive juice being especially formulated is used in combination, and to optimize young pig pancreas islet preparation method, improves pancreas islet yield.
Summary of the invention
The object of the present invention is to provide one kind in the case where not influencing islet function, can stablize and improve pancreas islet yield
Young porcine islet isolation method and for isolated digestive juice, this method is easily operated, reduces contamination probability.
A kind of youth's porcine islet isolation method, is digested by the way of shredding tissue, and digestive juice is with basis training
It supports base or balanced salt solution is that DNase I, neutral proteinase, I, III type is added on the basis of liquid phase is added to conventional digestion enzyme
Clostridiopetidase A, CaCl2, Trolox, growth hormone release inhibiting hormone.
5~10U/ml DNase I, 12000-4000u/g neutral proteinase, concentration are added in the digestive juice is
I, III Collagenase Type, the 1~10mM CaCl of 0.1~0.5mg/ml2, 1~10mM Trolox, 1~5ug/ml growth hormone release inhibiting hormone；Institute
The conventional digestion enzyme stated is V Collagenase Type, and addition concentration is 0.1~1mg/ml；The basal medium is RPMI1640,
The balanced salt solution is HBSS.
As a further improvement, the concentration of I, III Collagenase Type is 0.5mg/ml, CaCl in the digestive juice2For
1mM；Trolox is 5mM, growth hormone release inhibiting hormone 2.5ug/ml.
As a further improvement, shredding pancreatic tissue to tissue diameter is 1~5mm, and digestive juice is added to handle.
As a further improvement, liquid-solid ratio 20~50ml/g pancreas of digestive juice and pancreatic tissue.
As a further improvement, pancreas is first shredded roughly, handled with HBSS buffer, be further continued for using digestive juice after shredding
Processing.
As a further improvement, add 10ml buffer, it is 3~5mm that scissors, which shreds pancreatic tissue diameter, takes pancreas weight
After 2~5g, adds buffer to 50ml, mixes, 200rpm is centrifuged 1min, abandons 30~35ml of supernatant, is repeated once；It shreds in tissue
Add that continue after 25~30ml digestive juice to cut to tissue diameter be 1~5mm；Add digestive juice to 100ml in the tissue shredded.
As a further improvement, specifically includes the following steps:
1) the young pig pancreas of cold ischemia time < 30min is got out；
2) pancreas is successively oozed into the tincture of iodine, is transferred in sterile tray after physiological saline, pallet keeps ice bath；
3) lymph and connective tissue of pancreas are removed；
4) pancreas is transferred in 50ml sterile centrifugation tube and weighs and records；
5) plus 10ml buffer, scissors shred pancreatic tissue diameter as 3~5mm, take pancreas 2~5g of weight, add buffer
It to 50ml, mixes, 200g is centrifuged 1min, abandons 30~35ml of supernatant, is repeated once；
6) shredding in tissue plus continuing to cut after 25~30ml digestive juice to tissue diameter is 1~5mm；
7) tissue shredded is transferred in 250ml conical flask, adds digestive juice to 100ml；
8) taper bottle closure, 37 DEG C, 100rpm 15~20min of concussion, digestive juice add neutralizer to 250ml suspension when becoming muddy
Digestion；The neutralizer is the HBSS buffer containing 5% Swine serum；
9) 40 mesh metal screens are placed on beaker, filtration step 8) the tissue digestion liquid of preparation, and use 10ml
Syringe piston is ground on metal screen, until having filtered all digestive juices；
10) neutralizer cleans 2 times, and supernatant is abandoned in 200rpm, 2min centrifugation；
11) cold HBSS is resuspended, and 200rpm, 2min centrifugation are abandoned supernatant, is repeated 2 times；
12) precipitate, 200rpm, 2min centrifugation is resuspended in the maturation medium that pre-cooling is added；The maturation medium
For the RPMI1640 culture medium containing 10% Swine serum.
13) histocyte is resuspended in maturation medium, and average mark is into culture bottle, 37 DEG C, 5%CO2Culture, every pig point 4
The suspension blake bottle of~6 T-175.
Innovative point of the invention is as follows:
DNAse acts on extracellular dna, and extracellular dna plays a significant role the biofilm structure of most types, to biomembrane
Being formed has facilitation, and maturation with biomembrane is sticked in the starting that can influence biomembrane.DNAse can decompose mature biology
Film reduces the adhesion of biomembrane.Cause the reduction of matrix/extracellular polymeric intracellular to play a role the destruction of DNA, prevents
Released dna causes solution excessively viscous after only digesting.Conventional digestion enzyme hydrolyzes the proline polypeptide of cytoplasm, thus make cell from
It dissipates.
Neutral proteinase, can Hydrolysis of tissue albumen, be a kind of albumen for acting on protein peptide bond in neutral conditions
Protein can be hydrolyzed into polypeptide or free amino acid by enzyme, facilitate the discrete of cell, while can also effectively remove hydroxyl freedom
Base weakens the response to oxidative stress generated during proteolysis；I, III Collagenase Type acts on collagen Gly-Xaa-Yaa
Lysine/connection between arginine and glycine in (- Gly-) repetitive sequence cooperates V type glue from C-terminal hydrolytic collagen substance
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+Stable molecular conformation can be played with collagenase P KD in conjunction with the area CBD calcium binding sites, improve clostridiopetidase A
The purpose of stability can effectively improve digestive ferment utilization rate；Trolox can prevent oxidation caused by peroxynitrite
Stress, has anti-apoptotic, reduces the effect of oxidative stress, can effectively prevent generating oxyradical in digestion process to separation
Islet cells generate injury.Growth hormone release inhibiting hormone, which is added, can be effectively suppressed the exocrine function of pancreas, reduce tryptic activity, prevent
Only exocrine gland generates the characteristics of damaging action present invention combines various enzymes to islet cells, and anti-oxidation stress is used in combination, resists
Apoptosis, inhibition exocrine gland drug etc., are used in combination during separating islet cells, improve pancreas islet yield to reach, mention
High digestive ferment utilization rate reduces the purpose of islet cells separation economic cost.
In addition to the digestive juice being newly formulated that the present invention uses, while cooperating and being digested by the way of shredding tissue, it can
Obtaining yield is 1.67 × 104IEQ/g, the pancreas islet that purity is 75.3%, have conspicuousness relative to traditional separation pancreas islet method
Raising.Islet function testing result is shown, is 2.27 using its SI of islet cells that the present invention separates, 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 obtains:
1, Unit Weight pancreatic islet pick-up rate conspicuousness improves；
2, the case where successfully avoiding the generated cell bunchiness phenomenon under common digestion and excessively digesting, to reduce
Islet cells loss；
3, the pancreas islet separated is of moderate size, and meets the needs of big animal implant tests textured and clinical transplantation；
4, easily operated compared to common perfusion digestion method；
5, the utilization rate of digestive juice can be effectively improved using the present invention, reduced young porcine islet and separated economic cost.
Detailed description of the invention
Fig. 1 is various concentration I, III Collagenase Type cooperates V Collagenase Type to obtain islet cells under different digestions time
Efficiency and quality (100 ×)；
In Fig. 1 as a result, it was confirmed that in the case where concentration is 0.5mg/ml, separative efficiency is better than other groupings, therefore subsequent experimental is equal
Concentration is used to cooperate 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 various concentration I, III Collagenase Type cooperates V Collagenase Type under identical digestion time, cell
Apoptosis situation (100 ×)；
In Fig. 2 as a result, it was confirmed that relative to other two groups, concentration is under 0.1mg/ml and 0.5mg/ml, and Cell viability is preferable,
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 needed for cell is longer, increases the time of hot ischemic, is unfavorable for later period culture transplanting and uses.
DTZ dyes islet cells form (100 ×) after Fig. 3 is separation 48h；
In Fig. 3, experimental group is the isolated islet cells of the method for the present invention, and control group is poor compared with the method for the present invention
It is not that the V Collagenase Type islet cells isolated as digestive juice is used only.
Fig. 4 is that various concentration Trolox and growth hormone release inhibiting hormone are used cooperatively down, Intra-islet Apoptosis situation；
Fig. 4 is as the result is 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 Cell viability is preferable, from the aspect of economic cost, selects 5mM Trolox cooperation
2.5ug/ml growth hormone release inhibiting hormone uses.
Fig. 5 is islet cells yield；
In Fig. 5, experimental group is the isolated islet cells of the method for the present invention, and control group 1 is compared with the method for the present invention
The difference is that the V Collagenase Type islet cells isolated as digestive juice is used only, control group 2 is to be disappeared using tradition perfusion
The islet cells that change method cooperates V Collagenase Type isolated as 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 method for the present invention, and control group is poor compared with the method for the present invention
It is not that the V Collagenase Type islet cells isolated as digestive juice is used only.Existed using the islet cells that the present invention obtains
It is more suitable for later period transplanting needs in size.
Specific embodiment
It is intended to further illustrate the present invention with reference to embodiments, is not intended to limit the present invention.
Embodiment 1: the separation of young pig pancreas islet:
1, the containers such as the centrifuge tube to be used, conical flask, pallet are got out, and are marked；
2, young pig pancreas (cold ischemia time < 30min) is separated；
3, pancreas is successively oozed into the tincture of iodine, is transferred in sterile tray after physiological saline, pallet keeps ice bath；
4, removal pancreas extra lymph and connective tissue；
5, pancreas is transferred in 50ml sterile centrifugation tube and weighs and records；
6, plus 10ml buffer, after scissors cuts pancreas 1min, add buffer to 50ml, mix, 200g is centrifuged 1min, in abandoning
Clear 30~35ml, is repeated once；
7, it shreds in tissue plus continues to cut 10min to diameter is organized after 25~30ml digestive juice to be 1~5mm；
8, the tissue shredded is transferred in 250ml conical flask, adds digestive juice to 100ml；
9, taper bottle closure, 37 DEG C, 100rpm 15~20min of concussion, digestive juice add neutralizer to 250ml suspension when becoming muddy
Digestion；
10, metal screen is placed on beaker, 4~5ml/ times filtering, and with 10ml syringe piston on metal screen
It is ground, until having filtered all digestive juices (if pancreatic tissue is more on strainer should replace strainer in time)；
11, neutralizer cleaning strainer to total volume reaches 500ml (250ml+250ml), and supernatant is abandoned in 200g, 2min centrifugation；
12, cold HBSS is resuspended and merges precipitate, and 200g, 2min centrifugation are abandoned supernatant, is repeated 2 times；
13, precipitate, 200g, 2min centrifugation is resuspended in the maturation medium that pre-cooling is added；
14, histocyte is resuspended in maturation medium, and average mark is into culture bottle, 37 DEG C, 5%CO2(every pig is general for culture
The suspension blake bottle of 4~6 T-175 can be divided).
Embodiment 2: pancreas islet form, quantity detection
Material and reagent: 50ml centrifuge tube, 250ml centrifuge tube, 250ml conical flask, 500um metal screen, 10ml injection
Device, metal tray, water-bath, incubator, centrifuge tube shelf, scissors, haemostatic clamp, culture bottle, beaker, JPI complete medium, pig blood
Clearly, HBSS, antibiotic, the tincture of iodine, DNA enzymatic, physiological saline etc.
AO/EB detects Intra-islet Apoptosis: measuring acridine orange (AO), ethidium bromide (EB) each lmg, is dissolved in 10ml pH respectively
It is allowed to be made into the mother liquor of 100 μ g/ml in 7.2PBS, 4 DEG C are kept in dark place, are spare.With preceding mixed in equal amounts.100 μ l of cell suspension is taken,
4 μ l of AO/EB dyestuff is added to mix, takes one after another drop of in glass slide, fluorescence microscopy microscopic observation result.The result is shown in Figure 1,2,4.
Form, number and the purity of DTZ dyeing observation islet cells
10mg DTZ is dissolved in the dimethyl sulfoxide (DMSO) of 10ml, with after 0.22 μm of aperture membrane filtration degerming points
Dress is stored in -20 DEG C of refrigerators.When normal dyeing, every 1ml islet cells suspension is mixed with the DTZ storing liquid of 10 μ l, in 37 DEG C
It is incubated for microscopy after ten minutes, islet cells is dyed to the form of observation cell after scarlet, as a result sees Fig. 3.
Pancreas islet purity=DTZ stained positive islet cells rolls into a ball number/cell mass sum × 100%.The present invention obtains yield
It is 1.67 × 104IEQ/g, the pancreas islet that purity is 75.3%, have 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 detection
1, the function of insulin release test detection islet cells
When islet cells is cultivated respectively to the 6th day and the 10th day, the cell suspension natural sedimentation 6 of 6000IEQ is extracted respectively
Cell is sucked out after minute, is washed twice with the culture 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
It Jia Ru not 2ml low sugar containing 2.5mM culture solution (Hyclone culture medium) and 2ml the sugar culture solution containing 25mM high (Hyclone culture medium)
In in 6 hole culture dishes, it is put into 37 DEG C, 5%CO2After being further cultured for 2 hours in incubator, centrifuging and taking supernatant passes through chemiluminescence
Method detects insulin content.It calculates glucose stimulus index (SI).The function that SI > 1.8 represent islet cells is good.
Insulin release when glucose stimulus index (Stimulation index, SI)=25mM glucose culture/
2.5mM insulin release when glucose culture
1 each group islet cells glucose stimulus index of table
Experimental group is the islet cells that is prepared of the method for the present invention, control group be compared with the method for the present invention the difference is that
As a result the islet cells isolated as digestive juice using only V Collagenase Type illustrates that two groups of test results are not much different, into
One step, which illustrates the method for the present invention not, influences porcine islet function.
2, 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
A parallel hole is transferred to respectively in the centrifuge tube of 2ml, is detected wherein 1 part of every hole is separately added into 1ml Azol for insulin, separately
Outer 1 part does not add the cell suspension of Azol to detect for DNA.
The configuration of 2.1 reagents
1) configuration of Azol
RIA-BSA1.25g and acetic acid 57ml are dissolved in wiring solution-forming after mixing in the distilled water of 500ml, are put in 4 DEG C of refrigerators
Inside save backup.
2) configuration of FSA
By NaCl 4.383g, NaH2PO4·H2O 0.780g、NaH2PO4·2H2O 0.881g and RIA-BSA0.25g is molten
It is mixed in the distilled water of 500ml, wiring solution-forming, after the pH value of solution is adjusted to 7.35, is put in 4 DEG C of refrigerators and saves backup.
The detection of 2.2 insulin
The two hole cell suspensions of above-mentioned addition 1ml Azol are placed on ice, after being crushed 20 seconds with Ultrasonic Cell Disruptor, take 50 μ
L supernatant is dried in being put in oven in the centrifuge tube of 2ml, and 500 μ l FSA are added after drying, is put in -20 DEG C of refrigerators and is saved, most
Unify to detect insulin content by chemoluminescence method afterwards.
2.3DNA detection
The unified operation according to AxyPrep genomic DNA small volume of reagent box of the above-mentioned two hole cell suspensions for not adding Azol
Illustrate to extract DNA, specific steps are as follows:
1) deionized water or PBS suspension cell of 250 μ l is added.
2) the RNase A of 0.8 μ l is added, stands 1min at room temperature after shaking 15s.
3) the proteinase K of the buffer C-L and 8 μ l of 150 μ l is added, concussion 1min is uniformly mixed it, passes through
Then centrifuge tube is put in 56 DEG C of water-bath 10min by centrifugation.
4) the buffer P-D of 350 μ l is added, shakes 30s, 12,000 × g centrifugation using whirlpool to be uniformly mixed it
10min。
5) DNA is prepared pipe to be put among 2ml centrifuge tube, the mixed liquor immigration in previous step is prepared among pipe, 12,
000 × g is centrifuged 1min.
6) filtrate is discarded, is placed among the centrifuge tube of original 2ml pipe is prepared, the Buffer W1 of 500 μ l is then added
12,000 × g is centrifuged 1min.
7) filtrate is discarded, is placed among the centrifuge tube of original 2ml pipe is prepared, the Buffer W2 of 700 μ l is then added
12,000 × g is centrifuged 1min, and with identical method, the Buffer W2 that 700 μ l are added washed once again.
8) filtrate is discarded, is placed among the centrifuge tube of original 2ml pipe is prepared, 12,000 × g is centrifuged 1min.
9) among the centrifuge tube that DNA is prepared to the 1.5ml that pipe is put in another cleaning, 100- is added in the center for preparing periosteum
The Eluent or deionized water of 200 μ l, after standing 1min at room temperature, 12,000 × g is centrifuged 1min eluted dna.
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 value of 2 each group islet cells of table
Experimental group is the islet cells that is prepared of the method for the present invention, control group be compared with the method for the present invention the difference is that
As a result the islet cells isolated as digestive juice using only V Collagenase Type illustrates that two groups of test results are not much different, into
One step, which illustrates the method for the present invention not, influences porcine islet function.
Claims (3)
1. a kind of youth's porcine islet isolation method, which is characterized in that digested by the way of shredding tissue, digestive juice be
DNase I, neutral protein are added on the basis of being added to 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；
It is added 5 ~ 10U/ml DNase I, 4000-12000U/g neutral proteinase in the digestive juice, concentration is 0.1 ~
I, III Collagenase Type of 0.5mg/ml, 1 ~ 10 mM CaCl2, 1 ~ 10mM Trolox, 1 ~ 5ug/ml growth hormone release inhibiting hormone；Described is normal
Rule digestive ferment is V Collagenase Type, and addition concentration is 0.1 ~ 1mg/ml；The basal medium is RPMI1640, and described is flat
Weighing apparatus salting liquid is HBSS；
Specially following steps:
1) the young pig pancreas of cold ischemia time < 30min is oozed into the tincture of iodine, is transferred in sterile tray after physiological saline, pallet
Keep ice bath；
2) lymph and connective tissue of pancreas are removed；
3) pancreas is transferred in 50ml sterile centrifugation tube and weighs and records；
4) it is 3 ~ 5mm that plus 10ml buffer, scissors, which shreds pancreatic tissue diameter, takes pancreas 2 ~ 5g of weight, add buffer to 50ml,
It mixes, 200g is centrifuged 1min, abandons 30 ~ 35ml of supernatant, is repeated once；
5) shredding in tissue plus continuing to cut after 25 ~ 30ml digestive juice to tissue diameter is 1 ~ 5mm；
6) tissue shredded is transferred in 250ml conical flask, adds digestive juice to 100ml；
7) taper bottle closure, 37 DEG C, 100rpm 15 ~ 20min of concussion, digestive juice add neutralizer to stop to digest to 250ml when becoming muddy；
The neutralizer is the HBSS buffer containing 5% Swine serum；
8) 40 mesh metal screens are placed on beaker, filtration step 7) the tissue digestion liquid of preparation, and with 10ml syringe piston
It is ground on metal screen, until having filtered all digestive juices；
9) neutralizer cleans 2 times, and supernatant is abandoned in 200rpm, 2min centrifugation；
10) cold HBSS is resuspended, and 200rpm, 2min centrifugation are abandoned supernatant, is repeated 2 times；
11) precipitate, 200rpm, 2min centrifugation is resuspended in the maturation medium that pre-cooling is added；The maturation medium be containing
There is the RPMI1640 culture medium of 10% Swine serum；
12) histocyte is resuspended in maturation medium, and average mark is into culture bottle, and 37oC 、5%CO2Culture, 4 ~ 6 T- of every pig point
175 suspension blake bottle.
2. youth's porcine islet isolation method according to claim 1, which is characterized in that I, III type glue in the digestive juice
The concentration of protoenzyme is 0.5mg/ml, CaCl2For 1 mM；Trolox is 5mM, growth hormone release inhibiting hormone 2.5ug/ml.
3. a kind of youth's porcine islet isolation digestive juice, which is characterized in that digestive juice is with basal medium or balance salt
Solution is that DNase I, neutral proteinase, I, III Collagenase Type, CaCl is added on the basis of liquid phase is added to conventional digestion enzyme2,
Trolox, growth hormone release inhibiting hormone；5 ~ 10U/ml DNase I is added in the digestive juice, 4000-12000U/g neutral proteinase is dense
Degree is the I of 0.1 ~ 0.5mg/ml, III Collagenase Type, 1 ~ 10 mM CaCl2, 1 ~ 10mM Trolox, 1 ~ 5ug/ml growth suppression
Element；The conventional digestion enzyme is V Collagenase Type, and addition concentration is 0.1 ~ 1mg/ml；The basal medium is
RPMI1640, the balanced salt solution are HBSS.
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| 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 |
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| 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 |
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| 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 |
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