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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 observe the effect of over-expression of phospholipase Cβ1 (PLCβ1) on glucose-stimulated insulin secretion (GSIS). Methods The glucose concentration gradient was set at 10, 20, 40, 80 and 100 mmol / L, INS-1 cells were stimulated for 40 min, the insulin content in the cell culture supernatant was determined and the optimal glucose concentration was determined. 20, 40, 60, 80, 120min, with the optimal glucose concentration to stimulate, detect insulin content, determine the most appropriate stimulation time. ② The appropriate glucose concentration to stimulate INS-1 cells after a suitable time, RT-PCR detection PLCβ1 expression changes. ③ The PLCβ1 eukaryotic expression vector (PCMV-HA-PLCβ1) was constructed and transfected into INS-1 cells. The expression of PLCβ1 protein in INS-1 cells was detected by Western blot. ④ collect the transfected INS-1 cell culture supernatant to detect the insulin content. Results Insulin secretion was the highest in INS-1 cells stimulated with 40mmol / L glucose for 60min. The expression of PLCβ1 was significantly increased after stimulation with RT-PCR. The insulin content in INS-1 cell supernatant was (1.906 ± 0.080) ng / ml, which was significantly higher than that of control (0.740 ± 0.091) ng / ml transfected with PCMV-HA vector (P <0.01). Conclusion Overexpression of PLCβ1 significantly increased insulin secretion in INS-1 cells, suggesting that PLCβ1 may be involved in the transmission of GSIS.