成人睾丸支持细胞分离方法的建立及在胰岛移植中的应用  

Comparison of different isolation methods on adult Sertoli cells co-transplanted with islets

作　　者：李杨[1] 薛武军[1] 宋焕瑾[1] 冯新顺[1] 田晓辉[1] 丁小明[1] 宋勇[1] 丁晨光[1] 

机构地区：[1]西安交通大学医学院第一附属医院肾病中心肾移植科,陕西西安710061

出　　处：《细胞与分子免疫学杂志》2010年第6期552-555,559,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：陕西省"13115"科技创新工程重大科技专项计划资助项目(2007ZDKG-67);国家自然科学基金项目资助(30772096);卫生部临床重点学科资助项目;陕西省自然科学基金资助项目(30571799)

摘　　要：目的:建立成人睾丸支持细胞简便高效分离方法,并应用于胰岛移植。方法:A组采用胰蛋白酶、DNA酶、胶原酶、透明质酸酶一步消化法;B组采用胰蛋白酶和DNA酶第一步消化,胶原酶和透明质酸酶第二步消化;C组采用胰蛋白酶和DNA酶第一步消化,透明质酸酶第二步消化,胶原酶第三步消化。采用形态学观察和免疫组化鉴定支持细胞;MTT法和流式细胞术(FCM)测定3组支持细胞的活性和纯度;Western blot检测3组支持细胞Fas-L的表达;比较3组支持细胞与胰岛共移植至糖尿病鼠的效果。结果:经鉴定,分离的细胞具有支持细胞的特征;方法A和B分离的支持细胞数量较多,方法B和C分离的支持细胞杂质较少;MTT法检测结果显示,B组支持细胞活性显著高于A组和C组(P<0.05);Western blot结果显示,B组支持细胞Fas-L的表达水平显著高于A组和C组(P<0.05);FCM检测3种方法分离的S支持细胞纯度分别为(85.2±1.8)%、(92.3±2.5)%、(93.1±2.6)%,B组和C组的支持细胞纯度显著高于A组(P<0.05);将3组支持细胞与胰岛共移植至糖尿病鼠模型肾包膜下,B组胰岛移植物存活时间显著高于单独胰岛移植组和A、C两组(P<0.05)。结论:采用两步消化法能够获得纯度和活性较高的支持细胞,其与胰岛共移植能够显著延长移植物存活。AIM：To establish a more convenient and effective method for isolating adult Sertoli cells and apply the method to islet transplantation. METHODS： Trypsin, DNase, collagenase and hyaluronidase were used for a one-step digestion in group A. A two-step digestion was used in group B, in which testis tissues were first digested by trypsin and DNase and then were digested by collagenase and hyaluronidase. In group C, trypsin and DNase were used in the first step of digestion, hyaluronidase was used in the second step and collagenase was used in the third step. Sertoli cells were identified by morphology and immunohistochemistry and the viability and purity of Sertoli cells were detected by MTT and flow cytometry （FCM）. Expression of Fas-L was detected by Western blot and the effects of the co-transplantation of islets and Sertoli cells were compared between the three groups. RESULTS： Typical Sertoli cells were seen after isolation using the three different methods. Sertoli cells isolated by method A and method B were large in number while those isolated by method B and method C were pure. The results of MTT showed that the viability of Sertoli cells in group B was significantly higher than that in group A and group C （P〈0.05） and Western blot results showed that expression of Fas-L on Sertoli cells in group B was significantly stronger than that in group A and group C （P〈0.05）. The purity of Sertoli cells detected by FCM in group B and group C were significantly higher than that in group A （P〈0.05）. The survival of islets co-transplanted with Sertoli cells to renal capsule of diabetic mice was significantly longer in group B compared with that in control group as well as in group A and group C （P〈0.05）. CONCLUSION： Sertoli cells obtained by the two-step digestion method are of higher purity and viability, which significantly prolong the survival of islet graft by co-transplantation.

关 键 词：支持细胞 分离 胰岛 移植 

分 类 号：R392.11[医药卫生—免疫学]