磷脂酶cβ1的过表达对葡萄糖刺激胰岛素分泌的影响 - 第三军医大学学报.doc

磷脂酶Cβ1的过表达对葡萄糖刺激胰岛素分泌的影响 周恒宇1，邓华聪1，郑宏庭2，蒋 文3，南 静1，陈丹燕1 （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） 摘 要：目的　观察过表达磷脂酶Cβ1（phospholipase C β1,PLCβ1）对葡萄糖刺激胰岛素分泌（glucose-stimulated insulin secretion，GSIS）的影响。方法　①设定葡萄糖浓度梯度：10、20、40、80、100 mmol/L，分别刺激INS-1细胞40 min，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、120 min，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40 mmol/L葡萄糖刺激60 min，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。 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 中图法分类号： 文献标识码：A Overexpression of phospholipase cβ1 improves glucose-stimulated insulin secretion in INS cells ZHOU Heng-yu1, DENG Hua-cong1, ZHENG Hong-ting2, JIANG Wen3, NAN Jing1, CHEN Dan-yan 1 (1Department of Endocrinology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016; 2Department of Endocrinology, Xinqiao Hospital, Third Millitary Medical University, Chongqing 400037; 3Department of Emergency, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400010, China) Abstract: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 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 min respectively for the optimal time to induce insulin secretion. ② RT-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