共轭亚油酸对肝细胞胰岛素抵抗的影响

发布时间：2018-11-19 10:01

【摘要】：研究表明,共轭亚油酸(conjugated linoleic acid, CLA)对胰岛素抵抗的影响,可能存在异构体特异性,本文旨在阐明CLA对肝细胞胰岛素抵抗的影响及作用机制。采用高浓度胰岛素体外诱导法建立张氏肝细胞胰岛素抵抗模型,应用葡萄糖氧化酶-过氧化物酶(GOD-POD)法检测细胞葡萄糖消耗。细胞在含1%FBS和100nmol/L牛胰岛素的RPMI1640中孵育24h的条件下,葡萄糖消耗最少,即胰岛素抵抗最为明显。进一步通过模型持续时间的研究发现,建模36h后细胞仍存在葡萄糖摄取和利用障碍,且细胞形态没有明显的变化,即细胞模型在36h内具有稳定性。采用MTT法检测CLA对胰岛素抵抗细胞的毒性。结果表明：1,2,5和10μmol/L t10, c12-CLA或c9,t11-CLA孵育24h对胰岛素抵抗细胞的活力无显著影响。应用GOD-POD法检测1,2,5和10μmol/L CLA对胰岛素抵抗细胞葡萄糖消耗的改善是否存在异构体特异性。发现,t10, c12-CLA而不是c9, t11-CLA能够增加胰岛素抵抗细胞葡萄糖消耗,其作用呈明显的时间依赖性和剂量依赖性,其中适宜浓度为2μmol／L,使胰岛素抵抗细胞葡萄糖消耗增加17.29%(P0.001)；适宜时间为12h,使胰岛素抵抗细胞葡萄糖消耗增加4.82%(P0.01)。为深入探讨t10, c12-CLA改善细胞胰岛素抵抗机制,应用蛋白免疫印记法检测t10, c12-CLA对胰岛素抵抗细胞p-Akt蛋白表达水平的影响,发现1μmol/L和2μmol/L t10, c12-CLA处理12h能够增加胰岛素抵抗细胞p-Akt蛋白的表达。进一步应用磷脂酰肌醇3激酶(PI3K)抑制剂LY294002进行抑制实验,分析PI3K/Akt信号转导通路在t10, c12-CLA增强胰岛素抵抗细胞葡萄糖消耗中的作用。发现,PI3K抑制剂LY294002能够显著抑制t10, c12-CLA对胰岛素抵抗细胞葡萄糖消耗的改善作用,从而证明t10, c12-CLA对胰岛素抵抗张氏肝细胞葡萄糖摄取和利用的改善作用与其能够激活PI3K/Akt信号转导通路有关。综上所述,本文阐明了t10, c12-CLA能够通过激活PI3K/Akt信号转导通路改善肝细胞胰岛素抵抗。
[Abstract]:It is shown that the effect of co-linoleic acid (CLA) on insulin resistance may be specific, and the purpose of this study is to clarify the effect of CLA on insulin resistance and the mechanism of action. The insulin resistance model of human hepatocytes was established by the method of high-concentration insulin in vitro, and the glucose oxidase-peroxidase (GOD-POD) method was used to detect the cell glucose. The cells were consumed in RPMI1640 containing 1% FBS and 100nmol/ L of bovine insulin for 24 h, with minimal glucose consumption, i.e., insulin resistance Obviously, the study of the duration of the model found that the cells still had glucose uptake and utilization barrier after 36h of modeling, and there was no obvious change in the morphology of the cells, that is, the cell model was stable within 36h. Qualitative analysis of CLA on insulin resistance by MTT assay The results showed that 1, 2, 5 and 10. m u.mol/ L t10, c12-CLA or c9, t11-CLA were incubated for 24h to the activity of insulin resistance cells. No significant effect of CLA on glucose consumption of insulin-resistant cells by using GOD-POD method to detect 1, 2, 5 and 10. m u.mol/ L CLA It was found that t10, c12-CLA instead of c9, t11-CLA can increase insulin resistance to cell glucose consumption, and its effect is time-dependent and dose-dependent, where the appropriate concentration is 2.mu. mol/ L, and the insulin resistance to cell glucose consumption is increased by 17.29% (P0.001); the appropriate time is 12h, so that the insulin resistance cell glucose consumption is increased by 4.82 The effect of t10, c12-CLA on the expression level of p-Akt protein in insulin-resistant cells was detected by using the protein-immune-imprinting method. It was found that 1. mu.mol/ L and 2. m u.mol/ L t10, c12-CLA treated for 12h could increase the insulin resistance. The expression of p-Akt protein was studied. The inhibition of the expression of the P3K/ Akt signal transduction pathway in t10, c12-CLA was analyzed. It is found that the PI3K inhibitor LY294002 can significantly inhibit the improvement of the glucose consumption of t10, c12-CLA to insulin resistance cells, and it is shown that the improvement of t10, c12-CLA on glucose uptake and utilization of insulin-resistant Zhang's hepatocytes and its ability to activate PI3K/ The Akt signal transduction pathway is related. In conclusion, this paper states that t10, c12-CLA can be used to activate the PI3K/ Akt signal
【学位授予单位】：大连工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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