HepG2细胞胰岛素抵抗模型的建立与鉴定

Establishment and Identify of HepG2 Cells Model of Insulin Resistance

目的:采用胰岛素体外诱导法建立HepG2细胞胰岛素抵抗模型,为研究胰岛素抵抗及2型糖尿病提供一种可靠的体外胰岛素抵抗模型。方法:采用含不同胰岛素浓度的RPMI 1640培养基作用于HepG2细胞,葡萄糖-己糖激酶法检测作用不同时间点培养基中葡萄糖含量,MTT法检测各胰岛素浓度对HepG2细胞存活率的影响,确定胰岛素抵抗最佳作用浓度及时间;并以油红O染色观察细胞形态的变化;Real-time PCR法检测IRS-2 mRNA的表达,酶联免疫法检测葡萄糖-6-磷酸酶(G-6-Pase)的表达。结果:细胞产生胰岛素抵抗的最佳作用时间为36 h,最佳胰岛素浓度为1×10-8mol.L-1。建立模型后鉴定,模型细胞与正常细胞相比脂滴明显增多;IRS-2 mRNA的表达较正常细胞明显降低(P<0.05),G-6-Pase的表达较正常细胞明显升高(P<0.05)。结论:采用胰岛素体外诱导的方法,在胰岛素浓度为1×10-8mol.L-1,作用36 h后,可建立稳定的HepG2细胞胰岛素抵抗模型。

Objective：To establish the HepG2 cells model of insulin resistance by the method of insulin-induced in vitro,and provide a reliable model of insulin resistance in vitro for the study of the insulin resistance and the type 2 diabetes. Method： RPMI 1640 medium with different insulin concentrations was used to the HepG2 cells, the medium glucose content was detected at different time points by the method of glucose-hexokinase and the HepG2 cell viability of different insulin concentrations was detected by the method of MTT, and to determine the optimal concentration and time of the insulin resistance; then the changes of cell morphology was observed by the oil red O staining; the expression of insulin receptor substance（IRS）-2 mRNA was detected by the method of real-time PCR and the expression of G-6-Pase was detected by the method of ELISA. Result： The best time of insulin resistance was 36 h, the best concentration of insulin resistance was 1×10-8 mol·L-1. After the establishment of the model, the lipid droplets of model cells was significantly increased compared with normal cells; the IRS-2 mRNA expression was significantly reduced compared with normal cells （P＜0.05）; the G-6-Pase expression was significantly increased compared with normal cells （P＜0.05）. Conclusion： By the method of insulin-induced in vitro, a stable HepG2 insulin resistance model can be established when the 1×10-8mol·L-1 concentration of insulin act on the HepG2 cell for 36 h.