TLR2对骨骼肌线粒体生物发生及胰岛素敏感性的影响

发布时间：2018-04-26 05:14

本文选题：TLR2 + 脂肪酸　；参考：《河北医科大学》2017年硕士论文

【摘要】：近年来对胰岛素抵抗(insulin resistance,IR)的病因学研究成为一个热点,因为IR贯穿于高脂血症、糖尿病、高血压、痛风等多种代谢相关疾病,是这些疾病的共同土壤。众多研究资料表明炎症反应是IR发生的重要机制。从发现第一个与胰岛素抵抗相关的炎性细胞因子肿瘤坏死因子α(TNFα)以来,炎症机制已成为肥胖相关胰岛素抵抗的研究热点。肥胖状态下的炎症是一种低度的慢性炎症反应。TOLL样受体(Toll-like receptors,TLRs)作为模式识别受体不仅在感染免疫和损伤诱导的炎症反应中发挥重要作用,还参与代谢相关疾病的发生发展。TOLL样受体2(TLR2)是已克隆出的TOLL样受体家族中表达范围最广,识别病原微生物种类最多的成员。TLR2主要在哺乳动物的肺脏、心脏、大脑和肌肉组织中表达,其最突出的生物学功能就是直接或间接促进炎症因子的合成与释放。人类及啮齿类动物血浆葡萄糖60%~70%在骨骼肌代谢,骨骼肌是胰岛素刺激葡萄糖摄取的主要部位。脂代谢在骨骼肌胰岛素抵抗形成中扮演重要角色,线粒体是脂肪酸氧化的部位,表明线粒体功能在骨骼肌胰岛素抵抗形成中的重要地位。因此研究TLR2对骨骼肌线粒体生物发生、胰岛素抵抗的影响对代谢性疾病的防治具有重要的意义,为寻求糖尿病治疗新靶点提供科学依据。棕榈酸(Palmitate acid,PA)是游离脂肪酸中最主要的饱和脂肪酸,用其孵育可致细胞产生胰岛素抵抗,是建立体外细胞胰岛素抵抗模型的常用方法之一。本课题采用PA孵育L6成肌细胞构建胰岛素抵抗模型,并通过质粒上调和siRNA下调肌细胞中TLR2的表达,探究饱和脂肪酸是否是通过TLR2的表达影响骨骼肌线粒体生物发生,进而导致胰岛素抵抗。目的:研究TLR2对棕榈酸所致骨骼肌胰岛素抵抗的作用及其机制。方法:培养原始L6成肌细胞,使其分化成骨骼肌细胞,并用PA孵育,建立胰岛素抵抗模型。随后进行如下分组:对照组(control),棕榈酸组(PA),棕榈酸siRNA阴性对照组(PA+NC-siRNA),棕榈酸TLR2敲低组(PA+si-TLR2),棕榈酸空质粒组(PA+pcDNA),棕榈酸TLR2过表达组(pa+pcdna/tlr2)。通过rt-pcr及westernblot方法检测tlr2mrna及蛋白表达水平,确定转染成功后,用葡萄糖氧化酶法测定各组细胞培养基中葡萄糖浓度,评价胰岛素的敏感性。采用elisa方法测定六组培养基中炎症因子tnfα及il-1β的浓度。采用rt-pcr和westernblotting方法检测六组样本骨骼肌细胞中tlr2蛋白的表达水平,采用westernblotting方法检测六组样本骨骼肌细胞中线粒体生物发生相关蛋白pgc1α、mfn2、nrf-1以及胰岛素信号通路pi3k、p-akt、glut4蛋白的表达水平。多样本比较采用单因素方差分析,两样本间比较采用t检验。结果:1成功分化骨骼肌细胞分化组标志性分化基因desmin和myogenin的mrna表达较未分化组明显增加,差异具有统计学意义(p0.05);2胰岛素抵抗模型的建立成功分化的骨骼肌细胞,分为正常对照组和棕榈酸干预组。分别在12h、16h、20h、24h测定两组培养基葡萄糖浓度,在24h时,棕榈酸干预组葡萄糖浓度明显高于对照组,差异具有统计学意义(p0.05);3转染成功后六组样本葡萄糖浓度及炎症因子tnfα和il-1β的变化pa组培养基中葡萄糖浓度及炎症因子tnfα和il-1β较control组明显升高,差异具有统计学意义(p0.05);pa+nc-sirna组和pa+pcdna组较pa组葡萄糖浓度及炎症因子tnfα和il-1β无明显变化,差异无统计学意义(p0.05);pa+si-tlr2组较pa+nc-sirna组葡萄糖浓度及炎症因子tnfα和il-1β明显下降,差异具有统计学意义(p0.05);pa+pcdna/tlr2组较pa+pcdna组葡萄糖浓度及炎症因子tnfα和il-1β明显升高,差异具有统计学意义(p0.05);4转染成功后六组样本tlr2的表达与control组相比,pa组tlr2的表达明显升高,差异具有统计学意义(p0.05);pa+nc-sirna组和pa+pcdna组较pa组tlr2的表达无明显变化,差异无统计学意义(p0.05);PA+si-TLR2组较PA+NC-siRNA组TLR2的表达明显下降,差异具有统计学意义(P0.05),表明si-TLR2转染成功;PA+pcDNA/TLR2组较PA+pcDNA组TLR2的表达明显升高,差异具有统计学意义(P0.05)表明TLR2质粒转染成功;5转染成功后六组样本线粒体生物发生相关蛋白指标及胰岛素信号通路指标的变化与control组相比,PA组线粒体生物发生相关蛋白指标PGC1α、MFN2、NRF-1以及胰岛素信号通路PI3K、P-AKT、GLUT4的蛋白表达水平下降,差异具有统计学意义(P0.05);PA+NC-siRNA组和PA+pcDNA组较PA组线粒体生物发生相关蛋白指标PGC1α、MFN2、NRF-1以及胰岛素信号通路PI3K、P-AKT、GLUT4的蛋白表达水平无明显变化,差异无统计学意义(P0.05);PA+si-TLR2组较PA+NC-si RNA组线粒体生物发生相关蛋白指标PGC1α、MFN2、NRF-1以及胰岛素信号通路PI3K、P-AKT、GLUT4的蛋白表达水平升高,差异具有统计学意义(P0.05);PA+pcDNA/TLR2组较PA+pcDNA组线粒体生物发生相关蛋白指标PGC1α、MFN2、NRF-1以及胰岛素信号通路PI3K、P-AKT、GLUT4的蛋白表达水平下降,差异具有统计学意义(P0.05);结论:1高脂孵育可以诱导骨骼肌细胞胰岛素抵抗,表现为葡萄糖摄取减少,同时可使TLR2增加,炎症因子TNFα和IL-1β释放增加。2抑制或过表达TLR2可影响葡萄糖摄取,炎症因子分泌,线粒体生物发生相关蛋白PGC1α、MFN2、NRF-1及胰岛素信号通路PI3K、P-AKT、GLUT4蛋白的表达,说明TLR2表达增加损伤了线粒体生物发生相关蛋白,进而引起胰岛素抵抗。
[Abstract]:The etiological study of insulin resistance (insulin resistance, IR) has become a hot spot in recent years, because IR runs through many metabolic related diseases such as hyperlipidemia, diabetes, hypertension and gout, and is the common soil of these diseases. Many research data show that the inflammatory response is an important mechanism for the occurrence of IR. Since the anti related inflammatory cytokine tumor necrosis factor alpha (TNF alpha), the inflammatory mechanism has become a hot spot in obesity related insulin resistance. Inflammation in the state of obesity is a low degree chronic inflammatory response, the.TOLL like receptor (Toll-like receptors, TLRs) as a pattern recognition receptor not only in the infection of immune and injury induced inflammation. .TOLL like receptor 2 (TLR2), a member of the cloned TOLL like receptor family, is the most widely expressed, and the most widely recognized member of the pathogenic microorganism,.TLR2, is mainly expressed in the lungs, heart, brain and muscle tissue of mammalian, and its most prominent biological function. It is a direct or indirect promotion of the synthesis and release of inflammatory factors. Human and rodent plasma glucose 60%~70% is in skeletal muscle metabolism, the skeletal muscle is the main part of insulin stimulation of glucose uptake. Lipid metabolism plays an important role in the formation of insulin resistance in skeletal muscle. Mitochondria are the part of fatty acid oxidation, indicating mitochondrial work. It is important in the formation of insulin resistance in skeletal muscle. Therefore, the study of the mitochondrial biogenesis of skeletal muscle, the effect of insulin resistance on the prevention and control of metabolic diseases is of great significance, and provides a scientific basis for the search for new targets for the treatment of diabetes. Palmitic acid (Palmitate acid, PA) is the most important saturation in free fatty acids. Fatty acids, which can induce insulin resistance in cells, are one of the commonly used methods to establish insulin resistance models in vitro. This topic uses PA to incubate L6 myoblasts to construct insulin resistance model, and to explore whether saturated fatty acids are expressed through TLR2 expression through up-regulation and siRNA down regulation of TLR2 expression in myocytes. Mitochondrial biogenesis of skeletal muscle and insulin resistance. Objective: To study the effect and mechanism of TLR2 on the insulin resistance of skeletal muscle induced by palmitic acid. Methods: the primary L6 myoblasts were cultured to differentiate into skeletal muscle cells, and the insulin resistance model was incubated with PA. The following groups were grouped as follows: the control group (control), brown. Acid group (PA), palmitic acid siRNA negative control group (PA+NC-siRNA), palmitic acid TLR2 knockout group (PA+si-TLR2), palmitic acid empty plasmid group (PA+pcDNA), palmitic acid TLR2 overexpression group (pa+pcdna/tlr2). The expression of tlr2mrna and protein was detected by RT-PCR and Westernblot methods, and the cells of each group were determined by glucose oxidase method after the transfection was successful. The concentration of glucose in the medium was evaluated and the sensitivity of insulin was evaluated. The concentrations of inflammatory factors TNF a and IL-1 beta in the six groups were measured by ELISA method. The expression of TLR2 protein in the skeletal muscle cells of the six groups was detected by RT-PCR and westernblotting, and the midline of six groups of skeletal muscle cells was detected by westernblotting method. The expression level of PGC1 alpha, Mfn2, NRF-1, and insulin signaling pathway PI3K, p-Akt, GLUT4 protein in granular biogenetic protein. The diversity was compared with single factor analysis of variance, and t test was used for comparison between two samples. Results: 1 the expression of desmin and myogenin in the differentiation group of skeletal muscle cell differentiation was more than that of the undifferentiated group. The difference was statistically significant (P0.05); 2 insulin resistance model established the differentiated skeletal muscle cells, divided into the normal control group and the palmitic acid intervention group. The glucose concentration in two groups was measured in the 12h, 16h, 20h, 24h respectively. The glucose concentration in the palmitic acid intervention group was significantly higher than the control group at 24h, and the difference was statistically significant. Study significance (P0.05); 3 the glucose concentration and inflammatory factors TNF a and IL-1 beta in the six groups after the transfection, the glucose concentration and the inflammatory factor TNF alpha and IL-1 beta in the group PA were significantly higher than those in the control group, the difference was statistically significant (P0.05), and the concentration of glucose and the inflammatory factors in the pa+nc-sirna group and pa+ pcDNA group were compared with those of the PA group. There was no significant difference in beta (P0.05), but in group pa+si-tlr2, glucose concentration and inflammatory factors TNF a and IL-1 beta were significantly decreased, and the difference was statistically significant (P0.05); the glucose concentration and inflammatory factors in the pa+pcdna/tlr2 group were significantly higher than those in the pa+pcdna group, and the difference was statistically significant (P0.05); the 4 turn was 4. Compared with the control group, the expression of TLR2 was significantly higher in the group PA than that in the control group. The difference was statistically significant (P0.05), and there was no significant change in the expression of TLR2 in the pa+nc-sirna and pa+pcdna groups. The difference was statistically significant (P0.05), and the difference was statistically significant compared with that of the PA+NC-siRNA group. Significance (P0.05) showed that the transfection of si-TLR2 was successful, and the expression of TLR2 in group PA+pcDNA/TLR2 was significantly higher than that in group PA+pcDNA, and the difference was statistically significant (P0.05) indicated that the transfection of TLR2 plasmid was successful; 5 after the successful transfection, the changes of mitochondrial biogenesis related protein index and insulin signaling pathway index of 6 groups were compared with that of the control group, and the PA group mitochondria The protein expression levels of PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 were decreased, and the difference was statistically significant (P0.05); PA+NC-siRNA and PA+pcDNA groups were compared to PGC1 alpha, MFN2, and insulin signaling pathway. There was no significant difference in expression level and no significant difference (P0.05), and in group PA+si-TLR2, the mitochondrial biogenesis associated protein index of PA+NC-si RNA group, PGC1 alpha, MFN2, NRF-1, and insulin signaling pathway PI3K, P-AKT, GLUT4 protein expression level increased, and the difference was statistically significant (P0.05); PA+pcDNA/TLR2 group was more than the mitochondrial organisms. The protein expression levels of PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 were decreased, and the difference was statistically significant (P0.05). Conclusion: 1 high fat incubation can induce insulin resistance in skeletal muscle cells, showing a decrease in glucose uptake, increasing TLR2, and increasing the release of inflammatory factors TNF A and IL-1 beta. .2 inhibition or overexpression of TLR2 can affect glucose uptake, inflammatory factor secretion, mitochondrial biogenetic protein PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 protein expression, indicating that the increase of TLR2 expression damage the mitochondrial biogenetic protein, and then cause insulin resistance.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R58

【参考文献】