下调TCF7L2对胰岛素抵抗HepG2细胞胰岛素降解酶表达的调控作用观察

发布时间：2018-11-21 19:34

【摘要】：目的：探讨下调转录因子7类似物2(Transcription factor 7-like 2, TCF7L2)对胰岛素抵抗(Insulin resistance, IR) HepG2细胞胰岛素降解酶(Insulin degrading enzyme, IDE)表达的调控及可能机制。方法：细胞分组,将HepG2细胞分为空白组、TCF7L2干扰组、空载体组、IR组、IR+TCF7L2干扰组、IR+空载体组。采用高浓度胰岛素(5×10-6mol/L)持续作用24h诱导IR模型(IR-HepG2细胞)生成。以人TCF7L2 mRNA编码序列作为干扰靶点构建TCF7L2特异性小干扰RNA慢病毒载体(LV-TCF7L2-siRNA)转染空白组及IR组细胞,空载体病毒转染空载体组及IR+空载体组细胞。qRT-PCR法检测各组细胞TCF7L2及IDE mRNA的表达,Western blotting检测各组细胞TCF7L2、IDE、胰岛素刺激后蛋白激酶B (p-AKT)蛋白表达的变化,流式细胞术检测各组2-脱氧-D-葡萄糖(2-NBDG)荧光葡萄糖摄取率。结果：与空白组比较,IR组细胞葡萄糖消耗量及2-NBDG摄取率均明显降低(P0.01),证明IR细胞模型建立成功。qRT-PCR及Western blotting结果显示,R组TCF7L2及IDE mRNA及蛋白表达水平均明显低于空白组(P0.05),TCF7L2干扰组TCF7L2、IDE mRNA和蛋白表达水平较空白组、空载体组均明显下降,IR+TCF7L2干扰组TCF7L2、IDE mRNA和蛋白表达水平较IR组、IR+空载体组均明显下降(P0.05)。生理剂量胰岛素刺激后,IR组、IR+TCF7L2干扰组p-AKT蛋白水平较空白组明显下降(P0.01),各组总AKT水平差异无统计学差异(P0.05). TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较空白组和空载体组明显下降,IR+TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较IR组、IR+空载体组明显下降(P0.01)。结论：TCF7L2可通过调控IDE表达致肝细胞IR,其机制可能与减少胰岛素信号通路关键酶p-AKT蛋白表达有关。
[Abstract]:Aim: to investigate the regulation and possible mechanism of down-regulated transcription factor 7 analogue 2 (Transcription factor 7-like 2 (TCF7L2) on the expression of insulinase (Insulin degrading enzyme, IDE) in insulin-resistant (Insulin resistance, IR) HepG2 cells. Methods: HepG2 cells were divided into blank group, TCF7L2 interference group, empty carrier group, IR TCF7L2 interference group and IR empty carrier group. IR model (IR-HepG2 cells) was induced by high concentration of insulin (5 脳 10-6mol/L) for 24 h. TCF7L2 specific small interfering RNA lentivirus vector (LV-TCF7L2-siRNA) was constructed with human TCF7L2 mRNA coding sequence as the interference target, and transfected into blank group and IR group. The expression of TCF7L2 and IDE mRNA was detected by qRT-PCR method, and the expression of protein kinase B (p-AKT) protein after insulin stimulation of TCF7L2,IDE, was detected by, Western blotting. Fluorescence glucose uptake rate of 2-deoxy-D-glucose (2-NBDG) was detected by flow cytometry. Results: compared with the blank group, the glucose consumption and 2-NBDG uptake rate in IR group were significantly decreased (P0.01), which proved that the IR cell model was established successfully. The results of qRT-PCR and Western blotting showed that there was no significant difference between the two groups. The expression levels of TCF7L2, IDE mRNA and protein in group R were significantly lower than those in control group (P0.05). The expression level of TCF7L2,IDE mRNA and protein in TCF7L2 interference group was significantly lower than that in blank group, and the expression level of TCF7L2,IDE mRNA and protein in IR TCF7L2 interference group was significantly lower than that in IR group. IR empty carrier group were significantly decreased (P0.05). After insulin stimulation, the p-AKT protein level in IR group and IR TCF7L2 interference group was significantly lower than that in blank group (P0.01). There was no significant difference in total AKT level in each group (P0.05). The fluorescence glucose uptake rate of 2-NBDG in TCF7L2 interference group was significantly lower than that in blank group and empty carrier group, 2-NBDG fluorescence glucose uptake rate in IR TCF7L2 interference group was significantly lower than that in IR group, and that in IR empty carrier group was significantly lower (P0.01). Conclusion: TCF7L2 may regulate the expression of IDE and induce IR, in hepatocytes. The mechanism may be related to the reduction of the expression of p-AKT, a key enzyme of insulin signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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