环孢菌素A抑制大鼠胰岛素分泌的体外实验研究  

Cyclosporine A inhibits insulin secretion of rat islets in vitro

作　　者：孟树优[4] 刘倩[1,2,3] 孙富军[1,2,3] 汤云昭[1,2,3] 丁群[1,2,3] 孙茜[1,2,3] 张达[1,2,3] 李代清[1,2,3] 

机构地区：[1]天津医科大学代谢病医院,300070 [2]天津内分泌研究所,300070 [3]卫生部激素与发育重点实验室,300070 [4]西藏自治区人民医院

出　　处：《中华内分泌代谢杂志》2013年第4期339-343,共5页Chinese Journal of Endocrinology and Metabolism

基　　金：EFSD Grant Award for Collaborative Diabetes Research between China and Europesupported by Bristol-Myers-Squibbto Dai-QingLi;天津市应用与基础及前沿技术研究计划重点项目（08JCZDJC25100）;教育部留学回国人员科研启动基金;天津市重大科技攻关项目（中瑞合作,09ZCZDSF04500,胰岛素抵抗的检测、改善和机制）

摘　　要：目的 探索环孢菌素A抑制胰岛素分泌的分子机制。方法 经胆管不离体灌注胶原酶消化分离Wistar大鼠胰岛，环孢菌素A（0.5、1.0、2.5、5.0、10.0 μg/ml）干预不同时间后，吖啶橙/碘化丙锭染色，未经环孢菌素A处理的胰岛作为对照组，荧光显微镜下观察胰岛细胞存活变化。对照组和1.0 μg/ml环孢菌素A干预组中的胰岛分别孵育24 h后，进行高糖刺激胰岛素分泌实验；通过实时定量PCR方法探测P糖蛋白基因（abcb1b）、凋亡基因（casp3、Bcl-2）、胰岛素合成相关基因（pdx1、ins1、ins2）和胰高血糖素基因（glucagon）表达量的变化；通过罗丹明细胞内聚集实验评估P糖蛋白外排泵功能。结果通过胰岛存活状态的观察，确定1.0 μg/ml环孢菌素A为合适的干预浓度。环孢菌素A干预对体外胰岛高糖刺激下的第一时相分泌无抑制，而对第二时相抑制作用显著（P〈0.01）。环孢菌素A干预24 h后，abcb1b、ins1、ins2、pdx1、glucagon、casp3表达量均无变化；Bcl-2基因较对照组下调明显（P〈0.01）；环孢菌素A干预24 h后胰岛细胞内罗丹明聚集量的显著增加（P〈0.01）。结论 环孢菌素A干预抑制胰岛素第二相分泌，凋亡基因Bcl-2表达的变化是关键因素之一。胰岛素分泌的变化与P糖蛋白的表达、胰岛素合成及胰高血糖素无关，但是环孢菌素A对P糖蛋白功能的影响非常显著，不能排除P糖蛋白调节胰岛素分泌的可能性。Objective To explore the underlying mechanisms of inhibiting insulin secretion of rat islets by cyclosporine A in vitro. Methods Rat islets were isolated from pancreas by collagenase digestion. The islets were stained by acridine orange/propidium iodide and evaluated under fluorescence microscope after cyclosporine A were inoculated（0.5, 1.0, 2.5, 5.0, and 10.0 μg/ml）over different periods（6, 24, and 48 hours）. The islets treated only with the vehicle were served as control. After inoculation of 1 μg/ml cyclosporine A or the vehicle for 24 hours, insulin secretion of the islets was determined by radioimmunol assay（RIA）. The expressions of abcb1b, pdx1, ins1, ins2, glucagon, casp3, and Bcl-2 were evaluated by realtime fluorescence quantitative PCR after inoculations of cyclosporine A for 24 hours. A rhodamine 123 uptake measurement was used to analyze P-glycoprotein efflux pump function. Results Inoculation of 1.0 μg/ml cyclosporine A for 24 hours did not affect islet survival significantly. Only the second phase of insulin secretion was inhibited by the cyclosporine A inoculation（P〈0.01）, but not the first phase. Compared to the control group, the expressions of abcb1b, ins1, ins2, pdx1, glucagon, casp3 did not show any difference in the cyclosporine A inoculated group. But the expression of Bcl-2 was down-regulated significantly in the cyclosporine A inoculated group（P〈0.01）. The efflux pump function of P-glycoprotein was inhibited by the cyclosporine A inoculation（P〈0.01）. Conclusions Inhibitory effects of cyclosporine A on the second phase of insulin secretion may be through apoptosis pathway. Cyclosporine A did not influence biogenesis of insulin or glucagon. Even though cyclosporine A did not reduce the expression of P-glycoprotein, its specific inhibitory effect on P-glycoprotein in impairing insulin secretion could not be excluded. The underlying mechanism needs to be further investigated.

关 键 词：环孢菌素A 两相胰岛素分泌 胰岛Β细胞凋亡 P糖蛋白 

分 类 号：R587.1[医药卫生—内分泌]