CN105368771A - Mouse islet separation and purification method - Google Patents
Mouse islet separation and purification method Download PDFInfo
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- CN105368771A CN105368771A CN201510886141.2A CN201510886141A CN105368771A CN 105368771 A CN105368771 A CN 105368771A CN 201510886141 A CN201510886141 A CN 201510886141A CN 105368771 A CN105368771 A CN 105368771A
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Abstract
The invention discloses a mouse islet separation and purification method. A self-made puncture needle is adopted to reversely puncture a common bile duct and inject collagenase V to digest a pancreatic gland, and inslets are separated and purified by using the combination of density gradient medium centrifugation and manual sorting. Inset density is identified by dithizone staining, and inset activity is identified by AO/PI staining. Inset functionality is detected by a glucose stimulated insulin release experiment. By using the method to extract islets on a large scale, time can be greatly saved, the puncture success rate can be increased, and Ficoll400 formulation time and price can be reduced. In addition, all the yield, purity, activity and function of islets obtained by purification are relatively good, and the method is simple, fast, cheap and efficient.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of separation purification method of mouse islets.
Background technology
Along with expanding economy, the raising of the levels of substance of the people, the morbidity of diabetes (DiabetesMellitus, DM) is also growing.By the end of 2010, the morbidity of Chinese Adult DM reached 11.6%.But still lack the method for curing completely for diabetes at present, to the research of DM be also a lot of scientist in the world common faced by challenge.For diabetes B (Type2DiabetesMellitus, T2DM) fundamental research or type 1 diabetes (Type1DiabetesMellitus, T1DM) pancreatic islets transplantation depends on the research of animal insulin to a great extent, so successful isolated pancreatic islet, purifying then become the prerequisite of above-mentioned research.
Just there is scholar to extract pancreas islet from Guinea Pig Pancreas as far back as 1911, constantly have scholar to improve this method subsequently, occurred the method for the separation such as enzyme digestion, common bile duct perfusion, Ficoll400 density gradient centrifugation and purifying successively.But be confined to the difference of the operation system in each laboratory, the strain of mouse, separation and purification process, the yield of each laboratory pancreas islet, purity, vigor and islet function are also inconsistent.
Obtaining the pancreas islet that quantity is enough and function is good is the key that diabetes B fundamental research and type 1 diabetes pancreatic islets transplantation succeed.Now confirm, pancreas islet accounts for the 1%-2% of whole pancreas volume, and a mouse on average about has 2000 pancreas islet, and the pancreas islet extraction step of specification can extract about 200 pancreas islet.
And the output of mouse islets and function and all many-sides closely related.First relevant with the strain of mouse, the factor such as body weight and pancreas volume.The people such as Bock compare the pancreas islet content of 7 kinds of strain different weight mouse, find that the otherness contribution of the strain of mouse to pancreas islet quantity is maximum.In addition, the people such as Zhang Mei finds that mouse (normal mouse) the pancreas islet quantity of high body weight is significantly higher than under-weight mouse.
Secondly, the output of mouse islets and function are also relevant with separation method.As far back as 1911, the people such as Bensley just adopted manual selecting method to obtain pancreas islet and dye to it from cavy, but its pancreas islet obtained all can not be satisfactory from quantity or function.On the basis of this research, nineteen sixty-five, Moskalewski improved it, and have employed and shred the separation that the method adding enzymic digestion carries out pancreas islet, but under this method, collagenase can not fully arrive whole pancreas, the purity of gained pancreas islet is still poor.After 2 years, LacyandKostianovsky improves this method further, adopt common bile duct perfusion Hanks method fill pancreas, and full pancreas is shredded be placed in collagenase stir digestion.This is that the anatomical path that make use of animal itself first in the extraction of pancreas islet carrys out full pancreas.Subsequently, on the basis of this method the people such as Gotoh investigated common bile duct potting compound protoenzyme, Ficoll density gradient centrifugation method to extract pancreas islet.The islet yield that this method obtains, function than before several method are greatly improved, and this method is consuming time few, so this method is used till today always.But this method still has two point values to obtain us to note.One is that common bile duct venipuncture difficulty is comparatively large, and the requirement for operator is very high, and neophyty operation is failure very easily.The method of the people such as Gotoh also has a bit is worth us it is to be noted that Ficoll density gradient centrifugation, this method needs the Ficoll centrifugate (25% of preparation 4 kinds of different concns, 23%, 20.5%, 11%), the preparation steps very complicated of Ficoll, and the price of Ficoll is relatively high.The pancreas islet that this method obtains is mainly between this two-layer density medium of 1.069-1.096.And the osmotic pressure of Ficoll is a little more than Premeabilisation of cells pressure, to the cell in pancreas islet, there is harmful effect to a certain degree.
Except above-mentioned mouse itself and separation method are on except the impact of islet yield and purity.The kind of collagenase, purity, concentration, digestion time are also most important to the acquisition of pancreas islet.The people such as Liu Dingzhi compare LiberaseTL, CollagenaseP and CollagenaseV tri-kinds of different collagen enzymes to the impact of islet yield, find that the pancreas islet quantity using LiberaseTL digestion to obtain is maximum.The people such as Yesil find that the output of pancreas islet and the purity of collagenase present positive correlation.In addition using the collagenase of proper concn to be aided with suitable digestion time is also the important factor ensureing islet yield and purity.The people such as Zhong Jiaming adopt the collagenase P of different concns to digest different time to observe the output of pancreas islet, find at 1mg/ml, digest state the best that 25min obtains pancreas islet at 37 DEG C.
Summary of the invention
This research intend by homemade puncture needle and only a kind of density medium separation and purification is carried out to the pancreas islet of mouse, draw a kind of pancreas islet preparation method of simple and efficient, Cheap highly effective.
For achieving the above object, the present invention is by the following technical solutions:
A kind of mouse islets separation purification method, comprises the following steps:
(1) preparation of puncture needle: insulin syringe is cut off from 15U, scissors finishing incision position, take out a rifle head, spirit lamp burns one end that this rifle head connects with liquid-transfering gun, then rapid this rifle head to be coupled with the incision position of insulin syringe; Burn another rifle head, carry out reinforcing sealing with coupling place of its solute to above-mentioned rifle head and insulin syringe; Separately get a radicular vein transfusion needle, cut off by its syringe needle, then one end of intravenous infusion needle is connected the above-mentioned rifle head having burnt coupling, the other end connects a syringe, the needle slope finally carried by insulin syringe bending 60 ° upward;
(2) separation of mouse islets: the mouse blood of dead is fully drained, after spraying the alcohol disinfecting of 75%, row " V " type otch opens abdominal cavity, turn on the right side of mouse body, fully expose common bile duct and duodenum by jejunum and ileum and colon; At duodenum place ligation common bile duct, then puncture at the puncture needle that " V " font interface step (1) converged near liver common hepatic duct and cystic duct is obtained, puncture to press from both sides with bulldog clamp successfully and close puncture needle tail end common bile duct and avoid collagenase reflux; The collagenase V of precooling is poured into pancreas, makes it fully expand; Take off rapidly the pancreas poured into subsequently, be placed in the centrifuge tube containing collagenase V, static digestion 17min in 38 DEG C of constant water bath box; Mediate 3 times on multi-usage vortex mixer after digestion terminates, each 3 seconds; Subsequently, the Hanks balanced salt solution filling it up with precooling in centrifuge tube stops digestion; And on low speed centrifuge the centrifugal 2min of 1000rpm, rinse 2 times; Abandon supernatant after centrifugal, by resuspended for the Hanks balanced salt solution of precipitate precooling rear 40 order sterile metal screen filtrations, collect the tissue suspension after filtering;
(3) purifying of mouse islets: add after tissue suspension recentrifuge
and fully blow and beat suspendible with dropper; By tissue suspension good for suspendible centrifugal 20min under 2000rpm condition, after centrifugal end, pancreas islet is present in the middle of supernatant; Supernatant is transferred to centrifuge tube, and to add after the Hanks balanced salt solution of precooling centrifugal 5min under 1300rpm condition, after centrifugal end, pancreas islet is sunken at the bottom of pipe; Under pancreas islet being added Hanks balanced salt solution is resuspended and being placed on Stereo microscope, further manual selecting method carries out purifying; Finally the pancreas islet of purifying is moved into and cultivate containing in perfect medium.
In step (2), be that the collagenase V of 1mg/ml precooling pours into pancreas by 5ml concentration.
In step (2), the pancreas poured into is placed in containing 3ml concentration the centrifuge tube of the collagenase V being 1mg/ml.
In step (3), on average every mice pancreatic adds 7ml's
In step (3), described in
for preparing and passing through the lymphocyte separation medium filtered, 100ml's
in containing the urografic acid methylglucamine salt of 9.4g and the Ficoll of 6.2g, its density at 20 DEG C is between 1.077-1.080.
In step (3), described perfect medium is the RMPI1640 containing 15% foetal calf serum.
The invention has the beneficial effects as follows:
Adopt method of the present invention can save time significantly when large-scale extraction pancreas islet, improve puncture success rate, reduce time and the price of Ficoll400 preparation.Meanwhile, purifying gained islet yield, purity, vigor and islet function are all relatively good, are a kind of simple and fasts, the mouse islets separation method of Cheap highly effective.Be embodied in:
A kind of puncture needle of method design of the present invention, this pin convenient operation person grips, and its syringe needle size is 29G × 1/2 " (0.33mm × 12.7mm); its diameter is slightly less than the choledoch-diameter of mouse, can not wear out common bile duct easily when being easy to during puncture penetrate common bile duct and divide a word with a hyphen at the end of a line in common bile duct.In addition, after using another benefit of this puncture needle to be puncture failure, this pin is adopted to carry out the effect of regional perfusion fine.Adopt in the step of purifying
be actually and prepare and pass through the lymphocyte separation medium filtered, 100ml's
in containing the urografic acid methylglucamine salt of 9.4g and the Ficoll of 6.2g, its density at 20 DEG C is between 1.077-1.080, similar to pancreas islet density.And
osmotic pressure close with the osmotic pressure of islet cells, so islet function can be protected to a certain extent.Adopt in the present invention
separation and combination manual selecting method gained pancreas islet purity is significantly higher than simple manual selecting method, its mainly because
inherently a kind of purification process of pancreas islet, carry out after craft selects in the enterprising step in the basis of this method, the purity of pancreas islet can significantly improve.In the present invention
after being separated, the insulin secreting ability of gained pancreas islet also exists significant difference under high sugar and low sugar situation, points out the secreting function of its pancreas islet intact.
Accompanying drawing explanation
Fig. 1 is the structural representation of common bile duct puncture needle;
Fig. 2: different purification process is on the impact of islet yield, and CON represents simple manual selecting method purifying pancreas islet group,
representative adopts
density medium centrifugation is in conjunction with manual selecting method purifying pancreas islet group; Compared with control group, p=0.016;
Fig. 3: different purification process is on the impact of pancreas islet form, and CON represents simple manual selecting method purifying pancreas islet group,
representative adopts
density medium centrifugation is in conjunction with manual selecting method purifying pancreas islet group;
Fig. 4: the DTZ dyeing of separation and purification pancreas islet, CON represents simple manual selecting method purifying pancreas islet group,
representative adopts
density medium centrifugation is in conjunction with manual selecting method purifying pancreas islet group.What be wherein dyed to scarlet is islet cells group, and in yellow is nonislet cell group;
Fig. 5: the AO/PI dyeing of separation and purification pancreas islet, CON represents simple manual selecting method purifying pancreas islet group,
representative adopts
density medium centrifugation is in conjunction with manual selecting method purifying pancreas islet group.Wherein green fluorescence is viable cell in pancreas islet, and red fluorescence is apoptosis or dead cell in pancreas islet;
Fig. 6: different purification process is on the impact of islet secretion Regular Insulin ability.In control group, the insulin concentration under high sugar is compared with the insulin concentration under low sugar
*p=0.005; ?
discrete group, the insulin concentration #p=0.002 compared with the insulin concentration under low sugar under high sugar; In low sugar situation, adopt
the secretion capacity of separating obtained pancreas islet is lower than control group (p=0.029).
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
Embodiment
Material: laboratory animal is the 6-8 male ICR mouse in age in week of dead, and body weight 25-40g, purchased from Yangzhou University's comparative medicine center, feeds in Southeast China University's Experimental Animal Center.
Reagent: RMPI1640 substratum (gibco company), foetal calf serum (gibco company), collagenase V (Sigma company), dithizone (Chemical Reagent Co., Ltd., Sinopharm Group), apoptosis Acridine orange detection kit (triumphant base is biological), apoptosis PI staining kit (triumphant base is biological), penicillin and streptomycin (Hyclone company)
(LymphotcyteSeparationMedium, MP company of the U.S.), mouse islets element quantitative analysis enzyme-linked immunologic detecting kit (Shanghai skilful she bio tech ltd).
Laboratory apparatus: incubator (ThermoScientific company), Bechtop (Airtech company), thermostat water bath (Crystal company), phase microscope (CarlZeissJena company), low speed centrifuge (Zhong Jia branch office of Keda Innovation Co., Ltd), Stereo microscope (Shanghai Cai Kang opticinstrument company limited), fluorescent microscope (OLYMPUS, BX53) multi-usage vortex mixer (ScientificIndustries company).
Mouse islets separation purification method is:
Step 1, the preparation of puncture needle: insulin syringe 1 (adopting the aseptic insulin syringe of Shu Rui board single use in the present embodiment) is cut off from 15U, scissors finishing incision position; Take out a 0.1-10ul rifle head 2, spirit lamp burns one end that this 0.1-10ul rifle head 2 connects with liquid-transfering gun, then rapid this 0.1-10ul rifle head 2 to be coupled with the incision position of insulin syringe 1; Burn another 1-200ul rifle head, carry out reinforcing sealing with coupling place of its solute to above-mentioned 0.1-10ul rifle head 2 and insulin syringe 1; Separately get a disposable intravenous infusion needle 3, its syringe needle is cut off, one end connects the above-mentioned 0.1-10ul rifle head 2 having burnt coupling, and the other end connects a 5ml syringe 4, bending about 60 ° upward, syringe needle 5 inclined-plane finally carried by insulin syringe 1.The puncture needle structure be made into as shown in Figure 1.
Step 2: the separation of mouse islets: the mouse blood of dead is fully drained (digestion that blood can affect collagenase).Going after mouse being sprayed the alcohol disinfecting of 75% " " type otch opens abdominal cavity to V, turn on the right side of mouse body by jejunum and ileum and colon, fully exposes common bile duct and duodenum.At duodenum place ligation common bile duct, then puncture at " V " font interface converged near liver common hepatic duct and cystic duct, puncture to press from both sides with bulldog clamp successfully and close puncture needle tail end common bile duct and avoid collagenase reflux.The collagenase V (concentration is 1mg/ml) of 5ml precooling is poured into pancreas slowly, makes it fully expand.Take off rapidly the pancreas poured into subsequently, be placed in the centrifuge tube (15ml) containing 3ml collagenase V (concentration is 1mg/ml), static digestion 17min in 38 DEG C of constant water bath box.Mediate 3 times on multi-usage vortex mixer after digestion terminates, each 3 seconds.Subsequently, the Hanks balanced salt solution filling it up with precooling in centrifuge tube stops digestion.And on low speed centrifuge the centrifugal 2min of 1000rpm, rinse 2 times.Abandon supernatant after centrifugal, by resuspended for the Hanks balanced salt solution of precipitate precooling rear 40 order sterile metal screen filtrations, collect the tissue suspension after filtering.
Step 3, the purifying of mouse islets adds after tissue suspension recentrifuge
(on average every mice pancreatic adds
) and fully blow and beat suspendible with dropper.By tissue suspension good for suspendible centrifugal 20min under 2000rpm condition, after centrifugal end, pancreas islet is present in the middle of supernatant.Supernatant is transferred to 50ml centrifuge tube, the Hanks balanced salt solution adding precooling to 50ml under 1300rpm condition centrifugal 5min, after centrifugal end, pancreas islet is sunken at the bottom of pipe.Under pancreas islet being added 5mlHanks balanced salt solution is resuspended and being placed on Stereo microscope, further manual selecting method carries out purifying.Finally the pancreas islet of purifying is moved into and cultivate containing in perfect medium (RMPI1640 is containing the foetal calf serum of 15% mass content).
Step 4, the qualification of mouse islets: Purity: get 100ml dithizone powder dissolution in 10mlDMSO, 0.22 μm of aperture frit ,-20 DEG C keep in Dark Place.Face the used time to dilute with Hanks liquid 1:100, and add the DTZ dye liquor diluted after the pancreas islet of cultivation is changed liquid and to dye at 37 DEG C 15min.From containing repeated sampling the suspension of pancreas islet 3 times, each 200ul, drips in 3.5cm diameter Petri dishes, counts the cell mass number of red dye.The purity of pancreas islet is according to formula: pancreas islet purity=erythrophil group number/all cells group number × 100% calculates.Data represent with the form of mean ± standard deviation.Vigor is identified: get out sample diluting liquid, after the pancreas islet (100) cultivated is changed liquid, use 1ml sample diluting liquid resuspended, subsequently pancreas islet mixture is transferred to 1.5ml centrifuge tube, abandons supernatant after the centrifugal 5min of 2000rpm, it is resuspended to add 90 μ l sample diluting liquids, add AO (acridine orange respectively, AcridineOrange) dye liquor and each 5 μ l of PI (propidium iodide, Propidiumlodide) dye liquor, lucifuge dyeing 15min under room temperature condition.Subsequently pancreas islet is dripped on slide glass, observe under the condition of exciter filter wavelength 488nm and 536nm respectively under fluorescent microscope.Green Marker be viable cell, red-label be dead cell.
Step 5, the qualification of mouse islets function: the pancreas islet of separation and purification is inoculated in 24 orifice plates, 20, every hole pancreas islet, each sugared concentration arranges 3 multiple holes.After 37 DEG C of incubator overnight incubation, with the KRBB of sugar-free
[6](119mmol/LNaCl, 4.74mmol/LKCl, 2.54mmol/LCaCl
2, 1.19mmol/LMgCl
2, 1.19mmol/LKH
2pO
4, 25mmol/LNaHCO
310mmol/LHEPES, PH7.4, containing 0.5%BSA) after damping fluid rinse again with the KRBB damping fluid preincubate 0.5h containing 5mmol sugar, carry out hatching 2h respectively in the KRBB damping fluid (0.5mL) containing different sugar concentration with after the KRBB wash buffer of sugar-free again, supernatant is collected after end, by the concentration (specific experiment step operates according to the specification sheets of test kit, detects during actually operating by after diluted sample 40 times) of Regular Insulin in enzyme-linked immunosorbent assay supernatant.
Step 6, statistical analysis: adopt SPSS21.0 software to analyze.Experiment the data obtained represents with the form of mean ± standard deviation, and compare between group and adopt independent samples t test to compare, p<0.05 then thinks to have significant difference.
Result:
(1) output of mouse islets and form:
Adopt
separating obtained pancreas islet form is not of uniform size, and diameter is between 50-300 μm, and major part presents flat ellipse, and refractivity is good.Compared with simple manual selecting method (123.67 ± 9.61 pancreas islet), adopt
be separated every mouse and can obtain 95.33 ± 7.50 pancreas islet, output is a little less than manual selecting method, and difference has statistical significance (Fig. 2).Morphologically adopt
the integrity of separating obtained pancreas islet coating and smoothness are slightly worse than manual selecting method (Fig. 3).
(2) purity of mouse islets
Through separation and purification pancreas islet through DTZ dyeing after, pancreas islet is dyed to scarlet, but not pancreas islet agglomerate is not then colored (Fig. 4).Compared with simple manual selecting method (79.25 ± 5.50%), adopt
separating obtained pancreas islet purity on average about 92.57 ± 1.34%, its purity is significantly higher than simple manual selecting method, and difference has statistical significance (p=0.015).The pancreas islet of separation and purification is after AO/PI dyeing, and the viable cell in pancreas islet is caught green, and apoptosis or dead cell are dyed to redness (Fig. 5), simple manual selecting method and employing
separating obtained pancreas islet vigor does not have notable difference.
(3) secretion capacity of mouse islets
Adopt
separating obtained pancreas islet amount of insulin secretion under low sugar (2.8mmol/L) and height sugar (20mmol/L) stimulate is respectively 66.05 ± 13.30ng/ml and 256.08 ± 46.13ng/ml, insulin level under high sugar is 3.88 times under low sugar, and difference has statistical significance (p=0.002).And adopt simple manual selecting method gained pancreas islet amount of insulin secretion under low sugar (2.8mmol/L) and height sugar (20mmol/L) stimulate to be respectively 119.43 ± 24.27ng/ml and 226.70 ± 21.60ng/ml, insulin level under high sugar is 1.90 times under low sugar, and difference has statistical significance (p=0.005).Compared with simple manual selecting method, under low sugar concn
the insulin secreting ability of separating obtained pancreas islet is significantly lower than manual selecting method (119.43 ± 24.27ng/mlVS66.05 ± 13.30ng/ml, p=0.029); But, under high glucose concentration under two kinds of separation methods the secretion level of Regular Insulin but and indifference (226.70 ± 21.60ng/mVS256.08 ± 46.13ng/ml, p=0.374), concrete outcome is shown in Fig. 5.
Method totally about 80min consuming time in employing, many mouse are extracted pancreas islet simultaneously and can save time; This method every mouse on average can obtain pancreas islet 95.33 ± 7.50, purity 92.57 ± 1.34%, and vigor is indifference compared with manual selecting method; The pancreas islet that this method obtains under low sugar (2.8mmol/L) and height sugar (20mmol/L) stimulation in supernatant the level of Regular Insulin be respectively 66.05 ± 13.30ng/ml and 256.08 ± 46.13ng/ml (P=0.002).
Adopt this method can save time significantly when large-scale extraction pancreas islet, improve puncture success rate, reduce time and the price of Ficoll400 preparation.Meanwhile, purifying gained islet yield, purity, vigor and islet function are all relatively good, are a kind of simple and fasts, the mouse islets separation method of Cheap highly effective.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. a mouse islets separation purification method, is characterized in that: comprise the following steps:
(1) preparation of puncture needle: insulin syringe is cut off from 15U, scissors finishing incision position, take out a rifle head, spirit lamp burns one end that this rifle head connects with liquid-transfering gun, then rapid this rifle head to be coupled with the incision position of insulin syringe; Burn another rifle head, carry out reinforcing sealing with coupling place of its solute to above-mentioned rifle head and insulin syringe; Separately get a radicular vein transfusion needle, cut off by its syringe needle, then one end of intravenous infusion needle is connected the above-mentioned rifle head having burnt coupling, the other end connects a syringe, the needle slope finally carried by insulin syringe bending 60 ° upward;
(2) separation of mouse islets: the mouse blood of dead is fully drained, after spraying alcohol disinfecting, row " V " type otch opens abdominal cavity, turn on the right side of mouse body, fully expose common bile duct and duodenum by jejunum and ileum and colon; At duodenum place ligation common bile duct, then puncture at the puncture needle that " V " font interface step (1) converged near liver common hepatic duct and cystic duct is obtained, puncture to press from both sides with bulldog clamp successfully and close puncture needle tail end common bile duct and avoid collagenase reflux; The collagenase V of precooling is poured into pancreas, makes it fully expand; Take off rapidly the pancreas poured into subsequently, be placed in the centrifuge tube containing collagenase V, static digestion 17min in 38 DEG C of constant water bath box; Mediate 3 times on multi-usage vortex mixer after digestion terminates, each 3 seconds; Subsequently, the Hanks balanced salt solution filling it up with precooling in centrifuge tube stops digestion; And on low speed centrifuge the centrifugal 2min of 1000rpm, rinse 2 times; Abandon supernatant after centrifugal, by resuspended for the Hanks balanced salt solution of precipitate precooling rear 40 order sterile metal screen filtrations, collect the tissue suspension after filtering;
(3) purifying of mouse islets: add after tissue suspension recentrifuge
and fully blow and beat suspendible with dropper; By tissue suspension good for suspendible centrifugal 20min under 2000rpm condition, after centrifugal end, pancreas islet is present in the middle of supernatant; Supernatant is transferred to centrifuge tube, and to add after the Hanks balanced salt solution of precooling centrifugal 5min under 1300rpm condition, after centrifugal end, pancreas islet is sunken at the bottom of pipe; Under pancreas islet being added Hanks balanced salt solution is resuspended and being placed on Stereo microscope, further manual selecting method carries out purifying; Finally the pancreas islet of purifying is moved into and cultivate containing in perfect medium.
2. mouse islets separation purification method as claimed in claim 1, it is characterized in that: in step (2), is that the collagenase V of 1mg/ml precooling pours into pancreas by 5ml concentration.
3. mouse islets separation purification method as claimed in claim 1, it is characterized in that: in step (2), the pancreas poured into is placed in containing 3ml concentration the centrifuge tube of the collagenase V being 1mg/ml.
4. mouse islets separation purification method as claimed in claim 1, is characterized in that: in step (3), on average every mice pancreatic adds 7ml's
5. the mouse islets separation purification method as described in claim 1 or 4, is characterized in that: in step (3), described in
for preparing and passing through the lymphocyte separation medium filtered, 100ml's
in containing the urografic acid methylglucamine salt of 9.4g and the Ficoll of 6.2g, its density at 20 DEG C is between 1.077-1.080.
6. mouse islets separation purification method as claimed in claim 1, is characterized in that: in step (3), and described perfect medium is the RMPI1640 containing 15% foetal calf serum.
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| CN108130307A (en) * | 2018-02-08 | 2018-06-08 | 中山大学附属第医院 | A kind of Mice Islet Cells enrichment method of improvement |
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| CN113046305A (en) * | 2021-04-29 | 2021-06-29 | 佛山市第一人民医院（中山大学附属佛山医院） | Separation and purification method of islet cells |
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