短期胰岛素刺激对HIT-T15细胞胰岛素原基因表达的影响

摘要

目的：探讨短期外源性胰岛素刺激对HIT-T15胰岛β细胞胰岛素原（PI）基因表达的影响及机制。方法 HIT-T15仓鼠胰岛瘤细胞随机分为4组：含1.4 mmol/L葡萄糖完全培养基组作为空白对照(LG组)，为抑制内源性胰岛素释放干扰以低糖联合硝苯地平作为条件对照（LGC组），在LGC基础上分别加0.5 U/L或5 U/L的外源性胰岛素作为实验组(分别为LINS组和HINS组)，分别在刺激后0、30、60、90、120 min以荧光定量PCR检测各组PI mRNA，免疫组化检测各组细胞胰岛素受体底物1（IRS1）酪氨酸磷酸化水平。结果（1）LINS组、HINS组细胞PI mRNA表达均上调，在刺激后60 min PI mRNA表达量达高峰。（2）实验组在刺激后30 min IRS1酪氨酸磷酸化水平较对照组明显增高，高、低浓度胰岛素组细胞酪氨酸磷酸化达高峰时间不同。（3）LG组和LGC组间PI mRNA表达及IRS1酪氨酸磷酸化水平无明显差异。结论短期外源性胰岛素能上调胰岛β细胞PI基因的表达，且高浓度的胰岛素较低浓度胰岛素作用强。PI表达调控与IRS1信号传导有关。%Objective To investigate the short term effect of insulin on proinsulin gene expression of HIT-T15 insu-linnoma cells(pancreatic isletβ-cell). Methods The HIT-T15 cells were randomly divided into four groups.Blank Con-trol Group (LG):complete medium contain 1.4 mmol/L glucose. Control group (LGC):co-cultured nifedipine with medium in order to restain endogenous insulin release. Experimental group (LINS or HINS) add 0.5 U/L insulin or 5 U/L insulin on top of LGC. After being stimulated for 0, 30, 60, 90, 120 mins, proinsulin (PI) mRNA level were assessed by semi-quantitative RT-PCR. Insulin receptor substrate1 (IRS1) tyrosine phosphorylation was detected by immunocytochemistry. Results (1) Expression of PI was up regulated by both LINS and HINS, and peak at 60 mins. (2) After stimulation for 30 mins, the level of IRS1 tyrosine phosphorylation in the experimental group was significantly higher than control group, and the peak time be-tween LINS and HINS was different. (3) Between group of LG and LGC, the expression of PI mRNA and IRS1 tyrosine phos-phorylation show no difference. Conclusion Short term exogenous insulin stimulation can promote expression of proinsulin genes,which is concentration dependent. The expression and regulation of PI were related with IRS1 signal transduction.