miR-26b影响脂肪细胞胰岛素敏感性的机制分析

发布时间：2018-03-19 08:27

本文选题：miR-26b　切入点：脂肪细胞　出处：《蚌埠医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:阐明mi R-26b过表达对脂肪细胞胰岛素敏感性调控的分子机制。方法:1.高脂饮食喂养SD大鼠,建立饮食诱导肥胖(Diet-induced obesity,DIO)大鼠模型。高糖、高胰岛素环境下诱导建立3T3-L1/HPA-v胰岛素抵抗脂肪细胞模型;2.采用Real time PCR技术,检测动物及细胞模型中mi R-26b的表达;3.胰岛素(Insulin)、3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methyxanthine,MIX)、地塞米松(Dexamethasone,Dex)混合诱导剂诱导3T3-L1前体脂肪细胞分化为成熟脂肪细胞。胰岛素、3-异丁基-1-甲基黄嘌呤、地塞米松和罗格列酮(rosiglitazone)混合诱导剂诱导HPA-v前体脂肪细胞分化为成熟脂肪细胞;4.构建mi R-26b过表达慢病毒表达载体,感染人成熟脂肪细胞;48h后在荧光显微镜下,判断病毒感染的效率;5.采用同位素标记葡萄糖进行糖摄取实验,检测胰岛素刺激下脂肪细胞对葡萄糖摄取量的变化;6.Western Blot技术,检测PI3K信号通路中关键蛋白分子胰岛素受体(insulin recepor,IR)、胰岛素受体底物(insulin receptor substrate,IRS)、AKT、p-AKT、葡萄糖转运体-4(Glucose transporter type 4,GLUT4)的表达及GLUT4膜蛋白(m GLUT4)的含量;7.生物信息学预测mi R-26b的靶标,采用荧光素酶报告实验进行靶标验证。结果:1.高脂饮食诱导后SD大鼠出现肥胖及代谢紊乱的特征,成功构建DIO大鼠模型。DIO大鼠肠系膜脂肪组织中mi R-26b的表达水平下降,且与应用稳态模型评估的胰岛素抵抗指数(Homeostasis model assessment of insulin resistance,HOMA-IR)呈显著负相关;2.胰岛素抵抗细胞模型中mi R-26b的表达水平显著降低;3.mi R-26b过表达促进脂肪细胞胰岛素刺激下的葡萄糖摄取;4.mi R-26b过表达可以显著上调脂肪细胞胰岛素刺激下的m GLUT4含量;5.mi R-26b过表达可以显著提高磷脂酰肌醇激酶3(phosphatidylinositol3-kinase,PI3K)胰岛素信号通路中p-AKT水平;6.荧光素酶实验的结果表明,第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome 10,PTEN)为mi R-26b的直接靶基因。结论:mi R-26b可能通过调控PTEN的表达,影响PI3K胰岛素信号通路的正常传导,参与肥胖相关脂肪细胞胰岛素抵抗发生的分子机制。
[Abstract]:Objective: to elucidate the molecular mechanism of the overexpression of mi R-26b on insulin sensitivity of adipocytes. Methods: High-fat diet was fed to SD rats to establish Diet-induced obesity-induced diol (Dio) rat model. A 3T3-L1 / HPA-v insulin resistant adipocyte model was established under hyperinsulin-induced hyperinsulinemia. Real time PCR technique was used. The expression of mi R-26b was detected in animal and cell models. Insulin insulinator 3isobutyl-1-methyxanthine 3-isobutyl-1-methyxanthine (MIXX) and dexamethasone Dexen (Dexx) were used to induce the differentiation of 3T3-L1 precursor adipocytes into mature adipocytes. Insulin 3-isobutyl-1-methylxanthine was induced to induce the differentiation of 3T3-L1 precursor adipocytes into mature adipocytes. The mixture of dexamethasone and rosiglitazone induced HPA-v precursor adipocytes to differentiate into mature adipocytes. The vector of mi R-26b overexpression lentivirus was constructed and infected with human mature adipocytes for 48 h. To determine the efficiency of virus infection. Glucose uptake assay was performed with isotopic labeled glucose to detect the glucose uptake of adipocytes stimulated by insulin. 6. Western Blot technique was used to detect glucose uptake by adipocytes. To detect the expression of insulin receptor (insulin receptor), insulin receptor receptor (insulin receptor), insulin receptor substratesil, glucose transporter (-4Glucose transporter type 4GUT4) and the content of GLUT4 membrane protein (GLUT4) in PI3K signaling pathway. Bioinformatics was used to predict the target of mi R-26b. The target was verified by luciferase report experiment. Results: 1. The expression of miR-26b in mesenteric adipose tissue of DIO rats was successfully established, which was characterized by obesity and metabolic disorder in SD rats induced by high-fat diet, and the expression level of mi R-26b was decreased in mesenteric adipose tissue of DIO rats. There was a significant negative correlation between homeostasis model assessment of insulin resistance and HOMA-IR2.The expression level of mi R-26b in insulin resistance cell model was significantly lower than that in adipocytes stimulated by insulin. Glucose uptake 4.mi R-26b overexpression could significantly up-regulate the content of m GLUT4 in adipocytes stimulated by insulin. 5.mi R-26b overexpression could significantly increase the level of p-AKT in phosphatidylinositol3-kinase- PI3K) insulin signaling pathway. The results of luciferase assay showed that, Phosphatase and tensin homolog deleted on chromosome 10 PTENs are the direct target genes of mi R-26b. Conclusion the normal transduction of PI3K insulin signaling pathway may be affected by regulating PTEN expression. It is involved in the molecular mechanism of insulin resistance in adipocytes associated with obesity.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2

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