磷脂酶Cβ1过表达对葡萄糖刺激胰岛素分泌的影响  

Overexpression of phospholipase cβ1 improves glucose-stimulated insulin secretion in INS cells

作　　者：周恒宇[1] 邓华聪[1] 郑宏庭[2] 蒋文[3] 南静[1] 陈丹燕[1] 

机构地区：[1]重庆医科大学附属第一医院内分泌科,重庆400016 [2]第三军医大学新桥医院内分泌科,重庆400037 [3]重庆市中医院急诊科,重庆400010

出　　处：《第三军医大学学报》2009年第15期1471-1474,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30700384);重庆市自然科学基金(2006BB5098)~~

摘　　要：目的观察过表达磷脂酶Cβ1(phospholipase Cβ1,PLCβ1)对葡萄糖刺激胰岛素分泌(glucose-stimulated insulin secretion,GSIS)的影响。方法设定葡萄糖浓度梯度:10、20、40、80、100mmol/L,分别刺激INS-1细胞40min,检测细胞培养上清液中胰岛素含量,确定最适的葡萄糖刺激浓度;设定时间梯度:20、40、60、80、120min,以最适葡萄糖浓度刺激,检测胰岛素含量,确定最适的刺激时间。②以最适葡萄糖浓度刺激INS-1细胞适当时间后,RT-PCR检测PLCβ1表达变化。③构建PLCβ1真核表达载体(PCMV-HA-PLCβ1),转染INS-1细胞,Western blot检测INS-1细胞中PLCβ1蛋白的表达。④收集转染后INS-1细胞培养上清液,检测胰岛素含量。结果用40mmol/L葡萄糖刺激60min,INS-1细胞的胰岛素分泌量最大;RT-PCR观察刺激后PLCβ1表达显著升高;过表达PLCβ1的INS-1细胞培养上清液中胰岛素含量为(1.906±0.080)ng/ml,较转染PCMV-HA载体的对照组(0.740±0.091)ng/ml显著升高(P<0.01)。结论过表达PLCβ1显著增加INS-1细胞的胰岛素分泌,提示PLCβ1可能参与GSIS信息传递。Objective To investigate the role of phospholipase Cβ1 （PLCβ1） in the glucose-stimulated insulin secretion （GSIS）. Methods ①Glucose at the concentrations of 10, 20, 40, 80 mol/L and 100 mmol/L was used to treat INS-1 cells respectively for 40 min. The content of insulin in the INS-1 supernatant was detected for the optimal concentration of glucose. Then the decided concentration was used to the cells for 20, 40, 60, 80 and 120 rain respectively for the optimal time to induce insulin secretion. ②RT-PCR was used to detect the expression of PLCβ1 at the optimal glucose concentration for the optimal time. ③Eukaryotic expression vector PCMV-HA-PLCβ1 was constrcted and then transiently transfected into INS-1 cells under the optimal concentration and treatment time. The PLCβ1 protein was detected by Western blot analysis. ④Rat insulin ELISA kit was used to detect the content of insulin in the INS-1 supernatant. Results Exposed to INS-1 cells to 40 mmol/L glucose for 60 min, the content of insulin secretion reached the maximum and RT-PCR indicated the expression of PLβ1 was raised. Compared to the control [ （0. 740±0. 091 ） ng/ml], the insulin＇ s content in the supernatant of INS-1 cells with overexpressed PLCβ1 was raised [ （1. 906±0. 080） ng/ml] （P 〈0. 01）. Conclusion Transient overexpression of PLCβ1 in INS-1 cells improves the insulin secretion, so PLCβ1 probably participates in the signal transduction pathway of GSIS in INS-1 cells.

关 键 词：磷脂酶Cβ1 葡萄糖刺激胰岛素分泌 过表达 胰岛素 瞬时转染 

分 类 号：R335.6[医药卫生—人体生理学] R345[医药卫生—基础医学]