大鼠胰岛β细胞中mini-P糖蛋白的表达及其功能研究

The study of the expression and functions of the mini-P-glycoprotein in rat islet

目的探讨大鼠胰岛β细胞中65kDa的mini-P糖蛋白存在的可能性及其在两相胰岛素分泌中的作用。方法分别提取Wistar大鼠胰岛、胰腺及INS-1细胞中的总RNA,RT-PCR法检测多药耐药基因1(MDR1)的表达;萃取大鼠胰岛及INS-1细胞蛋白,用P糖蛋白特异性抗体C219行免疫印迹实验,检测P糖蛋白和mini-P糖蛋白的表达;体外胰岛刺激实验测定两相胰岛素分泌。结果在提取的大鼠胰岛、胰腺及INS-1细胞中均扩增出了MDR1基因。在胰岛匀浆蛋白中探测到65kDa的mini-P糖蛋白,在INS-1细胞蛋白萃取液中探测到了160kDa的P糖蛋白。在体外胰岛刺激实验中,P糖蛋白拮抗剂环孢菌素A抑制了胰岛素第二时相分泌,但第一时相分泌无明显变化。结论在INS-1细胞中有P糖蛋白的表达,在大鼠胰岛中有mini-P糖蛋白的表达,与胰岛素的两相分泌相关。

Objective It has been reported that a 65-kDa MDRI like protein （65-kDa protein） could function as sulfonylurea receptor and regulate biphasic insulin release. But the protein has not been further investigated. In this study, we wish to confirm existence and functions of the 65-kDa protein. Methods Total RNAs of rat kidney （positive control）, pancreas, islet and insulinoma cell line （INS-1） were extracted as templates and run with specific primers of MDR1. The PCP, products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS- PAGE system. P-glycoprotein was detected by specific monoclonal antibody to MDR1 （C219）. Bipbasic insulin release in vitro was measured in incubation medium by radioimmunoassay （RIA）. Results Compared with positive control, different fragments of MDR1 （RT-PCR products） localized in 3＇ end and 5＇ end, respectively, were obtained and analyzed by DNA sequencer. They were matched with MDR1 cDNA （99%）. In Western blot, a 65-kDa protein were detected in the islet homogenate, but not in that of INS-1 cells. The homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody （C219）. Insulin secreation of rat islet were stimulated by high glucose （16.7mmol/L）, and showed biphasie curve during 60min incubation. After inculation with cyclosporine A, a specific inhibitor of MDR1, the second phase of insulin secretion was reduced significantly but the first phase was not influenced. Conclusions The 65-kDa protein in rat islets is most likely a mini-P-glycoprotein. It may play a key role in regulating biphasic insulin release.