人源肝癌HepG2细胞胰岛素抵抗和逆转模型中FoxO1mRNA和蛋白表达水平及其意义

doi:10.13481/j.1671-587x.20170409

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (04): 709-714.doi: 10.13481/j.1671-587x.20170409

人源肝癌HepG2细胞胰岛素抵抗和逆转模型中FoxO1mRNA和蛋白表达水平及其意义

孙静¹, 魏虎来²

1. 兰州大学第二医院内分泌代谢科, 甘肃兰州 730000;
2. 兰州大学基础医学院医学实验中心, 甘肃兰州 730000

收稿日期:2016-11-08 出版日期:2017-07-28 发布日期:2017-08-01
通讯作者: 魏虎来,教授,博士研究生导师(Tel:0931-8915082,E-mail:weihulai@lzu.edu.cn) E-mail:weihulai@lzu.edu.cn
作者简介:孙静(1980-),女,黑龙江省哈尔滨市人,副主任医师,在读医学博士,主要从事糖尿病及糖尿病肾病基础和临床方面的研究。
基金资助:
中央高校基本科研业务费专项资金资助课题（861174）；甘肃省科技厅自然科学基金青年科研基金资助课题（1308RJYA065）；甘肃省卫计委卫生行业计划项目资助课题（GSWSKY-2015-64）

Insulin resistance of human hepatoma HepG2 cells and expression levels of FoxO1 mRNA and protein in reversal model and their significances

SUN Jing¹, WEI Hulai²

1. Department of Endocrinology and Metabolism, Second Hospital, Lanzhou University, Lanzhou 730000, China;
2. Center of Medical Laboratory, College of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China

Received:2016-11-08 Online:2017-07-28 Published:2017-08-01

摘要/Abstract

摘要： 目的：探讨人源肝癌HepG2细胞胰岛素抵抗（IR）模型（HepG2/IR）和逆转模型（HepG2/IR-PH）中叉头状转录因子O1（FoxO1）的mRNA和蛋白表达水平，阐明该基因参与IR的作用机制。方法：选择人源肝癌HepG2细胞，采用终浓度1×10^-10、1×10^-9、1×10^-8、1×10^-7、1×10^-6和1×10^-5mol·L^-1胰岛素诱导24、48和72 h，对照组细胞不加胰岛素，用葡萄糖氧化酶法检测上清液中葡萄糖水平，计算各组细胞的葡萄糖消耗量，以确定IR模型建立的最佳诱导条件；采用终浓度为0.156、0.313、0.625、1.250、2.500、5.000、10.000、20.000mmol·L^-1盐酸吡咯列酮（PH）诱导HepG2建立逆转抵抗模型，同时设立对照组，MTT法检测细胞增殖活性，确定PH的最佳逆转浓度；利用Real-time PCR法和Western blotting法检测各组细胞FoxO1 mRNA和蛋白表达水平。结果：1×10^-7 mol·L^-1胰岛素作用36 h，葡萄糖消耗量减少45.84%，发生明显的IR，与对照组比较差异有统计学意义（P<0.05）。与对照组比较，1.25 mmol·L^-1PH逆转抵抗24 h，细胞增殖活性无明显变化（P>0.05）。人源肝癌HepG2细胞发生IR时，与对照组比较，FoxO1 mRNA和蛋白的表达水平均明显升高（P<0.01）；IR模型逆转后，与对照组比较，FoxO1mRNA和蛋白表达水平差异无统计学意义（P>0.05）。结论：人源肝癌HepG2细胞IR与FoxO1mRNA的表达有关联性，FoxO1 mRNA表达水平检测有可能作为胰岛素增敏剂的药效评估指标，降低FoxO1 mRNA表达水平可作为2型糖尿病治疗的新靶点。

关键词: 胰岛素抵抗逆转, 叉头状转录因子O1基因, 人源肝癌HepG2细胞, 胰岛素抵抗

Abstract: Objective: To study the expression levels of forkhead transcription factor(FoxO1) mRNA and protein in the insulin resistance (IR) HepG2 cells model (HepG2/IR) and IR reversal HepG2 cells model (HepG2/IR-PH), and to explore its mechanism in IR.Methods: The HepG2/IR was induced with different doses of insulin (1×10^-10, 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6 and 1×10^-5 mol·L^-1) for different time(24, 36 and 48 h)in the HepG2 cells.The cells in control group were not treated with insulin. The glucose levels in supernant were determined by glucose oxidase method, and the glucose consumption in HepG2 in various groups were calculated to confirm the optimum induction conditions of HepG2/IR. The HepG2/IR-PH was induced with different doses of pioglitazone hydrochloride (PH) (0.156, 0.313, 0.625, 1.250, 2.500, 5.000, 10.000 and 20.000 mmol·L^-1) in the HepG2 cells, and control group was set up at the same time. The proliferation activities of cells were observed by MTT assay to confirm the optimum reversal concentration of PH. The FoxO1 mRNA and protein expression levels were detected by Real-time PCR and Western blotting methods.Results: The glucose consumption decreased by 45.84% in HepG2/IR after treated with 1×10^-7 mol·L^-1 insulin for 36 h, and there was significant difference compared with control group(P<0.05), the proliferation activity of cells had no change in the HepG2/IR-PH after treated with 1.25 mmol·L^-1 PH for 24 h(P>0.05).Compared with control group, the expression levels of FoxO1 mRNA and protein in HepG2/IR were significantly increased(P<0.01); compared with control group, the expression levels of FoxO1 mRNA and protein in HepG2/IR-PH had no significant differences(P>0.05).Conclusion: The IR of HepG2/IR is associated with the FoxO1 mRNA expression. The detection of FoxO1 mRNA seems to be an indicator to evaluate the efficacy of insulin sensitizer, and inhibiting the expression of FoxO1 mRNA may be developed as a potential therapy for type 2 diabetes.

Key words: insulin resistance, HepG2 cells, insulin resistance reversal, forkhead transcription factor

中图分类号:

R587.1

孙静, 魏虎来. 人源肝癌HepG2细胞胰岛素抵抗和逆转模型中FoxO1mRNA和蛋白表达水平及其意义[J]. 吉林大学学报(医学版), 2017, 43(04): 709-714.

SUN Jing, WEI Hulai. Insulin resistance of human hepatoma HepG2 cells and expression levels of FoxO1 mRNA and protein in reversal model and their significances[J]. Journal of Jilin University Medicine Edition, 2017, 43(04): 709-714.

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人源肝癌HepG2细胞胰岛素抵抗和逆转模型中FoxO1mRNA和蛋白表达水平及其意义

Insulin resistance of human hepatoma HepG2 cells and expression levels of FoxO1 mRNA and protein in reversal model and their significances

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