Wistar大鼠胰岛细胞体外分离_纯化及鉴定_英文__cropped

Wistar大鼠胰岛细胞体外分离_纯化及鉴定_英文__cropped 第 38 卷 第 3 期解剖学报 Vol138 ,No13 ACTA ANATOMICA SINICA 2007 年 6 月 J un. 2007 THE ISOL ATIO N , PURIFICATION AND ID ENTIFICATION OF WISTAR RAT’S ISL ET 3 TIAN Xiao2hong , BAI Shu2ling , TON G Hao ( )Institute of Tissue Engineering , College of B asic Medical Sciences , China Medical University , S henyang 110001 , China [ Abstract ] Objective The experiment aims at probing the best condition of the isolation and purification of rat islets. Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation. Then the purified islets were subjected to histological staining , electron microscopy and radioimmunoassay for identification of specificity and viability. Results The histological staining revealed that the viability and the purity of the purified islets were above 95 % and 85 % respectively. Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules. Radioimmunoassay demonstrated the secreted insulin concerntration between low2glucose groups and high2glucose groups varied significantly , which verified the good function of the islets. Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion. There are many factors that influence the quantity and quality of the acquired islets , such as the completed expansion of pancreas , the concentration and viability of collagenase and the digested time , etc . [ Key words ] Islect cell ; Isolate ; Purify ; Electron microscopy ; Radioimmunoassay ; Rat () [ CLC number ] Q813. 1 [ Document code ] A [ Article ID ] 052921356 2007032356 Wistar 大鼠胰岛细胞体外分离 、纯化及鉴定 3 佟浩田晓红 柏树令 ( )中国医科大学基础医学院组织 工程 路基工程安全技术交底工程项目施工成本控制工程量增项单年度零星工程技术标正投影法基本原理 学教研室 ,沈阳 110001 [ 摘要 ] 目的 探索 Wistar 大鼠胰岛分离纯化的最佳条件 。方法 采用肝胰管内灌注胶原酶消化分离胰岛 及 Ficoll 400 密度梯度离心纯化 。纯化后的胰岛经组织学染色 、电镜及放射免疫法鉴定其特异性和活力 。结果 组 织学染色显示纯化后胰岛的活力和纯度分别在 95 %和 85 %以上 。电镜显示纯化后的胰岛形态完整 ,包膜清晰 ,分 泌颗粒丰富 。放射免疫结果 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 明 ,低糖组和高糖组分泌胰岛素的浓度有显著差异 ,证明胰岛功能良好 。结论 肝 胰管内灌注胶原酶消化法是一种好的消化方法 。影响胰岛收获量的因素很多 ,例如胰腺的充分扩张 ,胶原酶的浓 度和活性以及消化的时间等 。 [ 关键词 ] 胰岛细胞 ; 分离 ; 纯化 ; 电镜 ; 放射免疫 ; 大鼠 isolated islets into 7 patients with type 1 diabetes mellitus In mid220 century , islet transplantation was and at the same time , the patients were administered with introduced into medical realm as a new method to treat nonglucocorticoid for immunosuppressive therapy. As a diabetes mellitus , which was expected to solve the result , the average time of good metabolism and dependence on insulin. People have explored it for many independence of insulin exceeded 12 months. But the years and gained some achievements. For example , 1 effect of clinical islet transplantation is not very satisfying Shapiro et al, who came from EdmontonResearch because of the inadequate quality and quantity of islet Center of Canada , obtained a good result about clinical graft as well as immunological rejection. If we could islet transplantation in 2000 . They injected the freshly transplant sufficient islets of good function and assist with immune depressant , the effect of clinical transplantation [ Received date ] 2006203216 [ Accepted date ] 2006210220 will be improved significantly. In the course of islets’ isolation and purification , the completed expansion of [ Foundation item ] This work was supported by grants from the and the viability of pancreas , the concentration Department of Education of Liaoning Province collagenase and the digested time are the most critical () 20122176. factors. This experiment adopted the common method of ) ( ) ( [ Biogra phy ] TIAN Xiao2hong 1980 —,female han, Shijiazhuang hepatopancreatic duct perfusing with collagenase and city , Hebei province , graduate student for a Master ’spurifying with Ficoll 400 discontinuous density gradient Degree . centrifugation , and explored the influencing factor which 3 To whom correspondence should be addressed were mentioned above . () E2mail : baishuling @hotmail . com. cn Tel : 0242325666625289 1 % osmic acid , then dehydrated by alcohol with different MATERIAL AND METHODS concentrations , dried , consperged with gold and observed ( ) by scanning electron microscopy J SM2T300 . Second , 1 . Animals Wistar rats , male or female , weighed 2502300g , according to the procedure mentioned above , when the purified islets were dehydrated , they were put into the were provided by the Animal Center of China Medical mixed liquor of absolute alcohol and oxidated propylene University , Shenyang , China . glycol , then sliced by ultramicrotome and stained by 2 . Isolation and purif ication of islets uranyl acetate and fructus lead , observed by transmission Wistar rats were anaesthed and sterilized after fasting () electron microscopy J EM1200 EX. for 12 hours. Then abdominal cavity was opened to expose the pancreas and hepatopancreatic duct . The liver , the 5 . The identif ication of islets’biological via bility The purified islets were cultured overnight in an duodenum and the stomach were moved out completely , incubator at 37 ?, 5 % COwith RPMI21640 culture 2 and the liver was turned over to head and covered with medium , which contained 10 % fetal bovine serum , wet gauze . The Vater ’s papilla was detected and the μ10mmolΠL glutamine , 100gΠL penicillin and 100mgΠL hepatopancreatic duct on the side of duodenum was streptomycin. The next day , the islets were cleaned with legated with silk. The other end of the hepatopancreatic Hanks solution and selected under a inverted microscope . duct , near the hepatic portal , should be ligated as well . Fifty islets were counted as a group and stimulated with The heart should be cut to sacrifice . The scalp ( ) KRHB Krebs2Ringer2HEPES2bicarbonate solution of acupuncture of 415 or 515 , which was linked with the ( ) low2glucose 218mmolΠL for 4 hours. After that , the injector containing type V collagenase solution , was used islets were centrifuged at 161g , 4 ?, for 3 minutes ; then to insert into the hepatopancreatic duct from the side of the supernatant was collected and stored at 220 ?. Then ( ) duodenum Fig. 1 and Fig. 3 . When expanded the same islets were cleaned and stimulated with KRHB completely , the pancreas was taken out promptly , put into solution for 4 hours and the supernatant was collected 50ml centrifuge tube , and then digested in water bath at afterwards as well . The supernatant was determined by 38 ? for 10215minutes. After that , the centrifuge tube radioimmunoassay. KRHB solution was prepared as was taken out and shaken violently until the content was ( ) follows : NaCl 140mmolΠL mm, KCl 316mm , CaCl 2sand2shaped. Then the digestion was ceased immediately 110mm , MgSO015mm , NaHPO015mm , NaHCO 4 2 4 3with a large amount of cold Hanks solution which () 210mm , HEPES 10mm p H714, glucose 3mm. contained 10 % fetal bovine serum. Next , the content was screened through the metallic filter net of 40 mesh into RESULTS another 50 ml centrifuge tube followed by centrifuging with 161g for 3 minutes at 4 ?. The supernatant was 1 . The biological observation of islets by a light dumped and new Hanks solution was added for another microscope centrifugation . The same centrifugation was repeated twice The pancreas expanded immediately and presented in order to make the islet clean. At last , the deposition red bubble2form after perfused with collagenase . There was transferred into another 15ml centrifuge tube , added were few blue cells in the purified islets after stained with 25 % Ficoll and mixed gently and sufficiently. Then trypan blue and the viability was above 95 %. Most of the 23 % , 20 % and 11 % Ficoll was added carefully in islets were red with round or oval shapes and different order , centrifuged with 1 450g for 20 minutes at 4 ?. μμ( ) diameters from 50m to 300m Fig. 2after stained with After that , the purified islets were collected at the layer of DTZ. The purified islets could be distinguished easily 11 %220 % and 20 %223 % and cleaned 3 times with because the external secreting tissue was not stained and Hanks solution. the purity was about 85 %. The purified islets were of islets’via bility ,3 . The evaluation specif icity and ( ) ( ) obtained about 4002500IE insulin equivalent from a purity Wistar rat at most . The viability of islets was judged by trypan blue 2 . The ultra structure of islets staining: the islets ’suspension 011ml was added with From scanning electron microscopy , the islets Hanks solution 018ml and 014 % trypan blue solution existed in the form of clusters and the majority of them 011ml ,then mixed well and counted within 5 minutes at β cells. The latter had integrity connective tissue ware room temperature . membrane and all kinds of“secreting holes”, excavation The specificity of islets was identified by dithizone ( ) β and granules on the surface Fig. 4. The cells were ( ) DTZ staining : absolute alcohol was used for the located in the center of islets and had numerous round (β ) secreting granules granulesobserved by transmission preparation of DTZ. The method was as follows : 10mg of ( electron microscopy. Besides , there were a few DTZ and 3ml of absolute alcohol containing 1167 % μ) NaOH 50l and 12ml of Hanks solution were sterilized mitochondria , Golgi complex and endocytoplasmic μwith 0122m filter membrane and stored at 220 ?. It β reticulum incells and lots of different nuclear cores in ( ) should be diluted with Hanks solution p H718 at a proportion of 1?100 when it was used. Ten minutes after β the granules , in the shape of round , rectangle , staining at 37 ?, the quantity of islets and external aciculiform and so on , which were different from those in α cells and the gap between the membrane and the core secreting tissue could be counted by inverted microscope , ( ) α was wider Fig. 5 . By contrast , cells had many and the ratio of staining cells and external secreting cells secreting granules with round cores which were on the side could be calculated. ( ) β of membrane Fig. 6 . granules are the special cell 4 . The observation of islets’ultra structure β organ ofcells and they are the source of insulin. First , the purified islets were fixed with 215 % glutaraldehyde , washed by cacodylate buffer , fixed with Vol . 38 ,No . 3 ’s isletThe isolation , purification and identification of Wistar ratTIAN Xiao2hong et al . 〃357 〃 Fig. 1 The distended pancreas after collagenase perfusing Fig. 2 The ideograph of the operation ( ) Fig. 1 , Fig. 2Note : 1 , pancreas ; 2 , stomach ; 3 , liver ; 4 , duodenum ; 5 , spleen arrow a , the porta hepatic end of hepatopancreatic duct arrow b , the duodenum end of hepatopancreatic duct arrow c , the hepatopancreatic duct Fig. 3 The light microscopic photograph of islet cells after dithizone staining. ×200 Note :arrows demonstrating the secreting holes βFig. 4 The scanning of electron microscopic photograph of islet cells after fixing. ×5 000 Fig. 5 The ultrastructure of cell . ×8 000 βNote : arrows demonstrating nuclear cores ofgranules αFig. 6 The ultrastructure of cell . ×8 000 Note : arrows demonstrating the nuclear cores of secreting granules Table The result of glucose2stimulating test The results of glucose2stimulating 3 . test by ra dioimmunoa ssay ()the concertration of insulin mIUΠL No . The purified islets were stimulated by low2glucose low2glucose groups high2glucose groups and high2glucose KRHB solution and the concentrations of 21191 128 821 () ()insulin were 25160 ?2130mIUΠL and 92177 ?7191 25164 91190 2 ( ) mIUΠL respectively Table. The latter was as 316 times 27154 92197 3 as the former , which showed that the purified islets were 28130 87135 4 sensitive to glucose2stimulating and had good functions. 24147 105125 5 25175 96188 6 DISCUSSION ( ) x ? ?s 25160 ?2130 92177 ?7191 P ?01001 One of the major problems in clinical islet transpl2 changed into sand2form , then it was filtered and the antation for type 1 diabetes mellitus is the insufficiency of 2 digestion was stopped immediately or the floccule would . How to obtain islets of quantity and viability of islets be formed and wrapped the islet . Because the collagenase high quantity and viability is one of major concerns in 3 has no special inhibitor , we have to stop digesting by research nowadays. Since Moskalewskisinvented the ( ) ( ) diluting add much Hanks solution, cooling ice bathmethod of collagenase2digesting , many researchers have ( ) and adding protein serum. And last , there was a serious adopted different approaches to explore and develop in loss of islets in the course of purification for the islets this field of study. Although there have been some improvements , yet the results haven′t been very might adhere to other tissue and its density would be satisfying. There are two methods commonly used to changed. So the deposition should be mixed well with isolate islets : one is cutting the pancreas to small scrap s 25 % Ficoll and well2distributed. In addition , all the for digestion ; the other is perfusing collagenase or Hanks centrifuging course should be processed at a low solution into the hepatopancreatic duct . It was reported temperature to maintain the viability of cells. that the latter was better than the former because the In the experiment , many anthropic factors affected hepatopancreatic duct perfusing might expand exocrine the results : from the initial mechanical disruption to static 4 gland acinus and play a role of initial digesting. digestion , then to the application of continuous digesting 9 Besides , there are many factors that affect the islet ’s equipment for the isolation of islets by Camillo Ricordi harvest , such as the concerntration and viability of et al . Since the invention of the auto2isolating system 10 collagenases and digesting time and so on. Type V or P () controlled by computer Lakeyet al,the technique for collagenase are frequently used and the concerntration is isolating and purifying islets has been improved gradually () 0132210 gΠL . A report said that the combination of and has the tendency of standardization and automation , 5 different digestive enzymes could improve the result. but there is a shortage of correlated practice . So there is a But it is believed that collagenase has a high viability in lot to do to improve the method and techniques to acquire 2 + the condition of Caexisting , 38 ? and p H7 . 8 . If the higher a extraction rate of islets , which is the foundation digesting time is short , the islet can’t be isolated from to bring benefits to the patients with diabetes mellitus. pancreas completely and the density of the islet will be REFERENCE changed which will lead to islet loss in the course of 1 ] Shapiro AM ,Lakey J R , Ryan EA , et al . Islet transplantation in seven purification. On the contrary , if the digesting time is patients with type 1 diabetes mellitus using a glucocorticoid2free long , the islet can be wrapped by the dope and it will be ( ) immunosuppressive regimen J . New Eng J Med , 2000 , 343 4 : 2302 difficult to isolate the islet . As to purification , Ficoll 400 238 . Mathieu C. Current limitations of islet transplantation J . Transplant 2 discontinuous density gradient is commonly used. But it is () Proc ,2001 ,33 122:1707 . expensive and the course of preparation , sterilization and ] Chen CHQ , Zhan WH , Wang J P , et al . Experimental comparisons of 3 density2forming is complex , in addition to its toxicity to several methods for isolating and purifying of rat islets of langerhansJ . () Acad J SUMS ,2002 ,23 2:1182120 . 6 74 ] Zhou MH , Chen DM , Tang JM , et al . Preparation of rat islet transplant islets, so someone suggested using Dextranor 8 () and xenotransplantationJ . Chin J Organ Transplant ,1998 ,19 4:2002 Histopaque21077instead.201 . The pancreas is a complex secreting gland and the ] Zhou WX , Cui YF , Yan ZHQ ,et al . The research on isolating result of 5 adult2pig islet with simple enzyme and complex phosphoesterasum J . islet only occupies 1 %22 % of it , so it is difficult to () Chin J Exp Surg ,1999 ,16 6:569 . isolate and purify islets. There are many factors that affect Salvalaggio PR , Deng S , Ariyan CE , et al . Islet filtration : a single and 6 the result , so a little of negligence may result in“non2 rapid new purification procedure that avoids ficoll and improves islet islet ”. Some experience is as follows : First , the perfusing () mass and functionJ . Transplantation ,2002 ,74 6:8772879 . of the collagenase must be correct and full . In this ] Wang HX , Guo SHL , Lin Y , et al . The experimental study of two 7 course , the spleen should be raised to ensure the end of different methods for isolating and purifying of rat islet J . Chin J Exp () Anim ,2002 ,12 5:3092311 . pancreas which was linked to the spleen was also Liu M , Shapiro ME. A new method for isolation of murine islets with 8 expanded. If the cannula was not in the right place , it ( ) markedly improved yields J . Transplant Proc , 1995 , 27 6 : 32082 might lead to the destruction of the pancreas acinar and 3210 . the isolation of islet wouldn′t be complete . Second , the Ricordi C ,Lacy PE , Scharp DW. Automated islet isolation from human 9 ( ) pancreasJ . Diabetes ,1989 ,38 Suppl 1:1402142 . collagenase solution should be prepared on the spot . In 10 Lakey J RT ,Warnock GL ,Brierton M ,et al . Development of an automated the course of digestion , the solution was acidified computer2controlled islet isolation systemJ . Cell transplantation ,1997 , gradually , so HEPES was added which has a strong buffer () 6 1:47257 . to maintain the p H value . Third , after digestion , the ( )编辑 张艳 郭崇洁 centrifuge tube should be shaken strongly until the tissue