TLR4在脂肪酸诱导人骨骼肌细胞胰岛素抵抗中作用的研究

发布时间：2018-05-15 01:04

本文选题：代谢综合征 + 脂肪酸　；参考：《第四军医大学》2012年硕士论文

【摘要】：研究目的：代谢性疾病是一个世界范围的问题，严重地影响着人民的生活质量。代谢性疾病的发生与长期能量摄入正平衡引起的肥胖有关。肥胖在导致其他代谢性疾病时，一个重要的环节是全身胰岛素抵抗的形成。因此，胰岛素抵抗是代谢性疾病形成的关键。脂肪组织等形成胰岛素抵抗的主要诱导因素是游离脂肪酸增加；游离脂肪酸的增加则与长期能量正平衡导致的肥胖有关。肥胖实质是一种低度炎症反应。但是肥胖致炎症反应的发生机制尚不明确。人体的免疫系统是受到精密调控的，炎症反应的过程往往有一个或几个关键调节点，已有研究显示TLR4可能是肥胖致低度炎症反应的一个关键调节点。TLR4在炎症因子基因启动和转录调控过程中占有重要的地位，同样在肥胖所致炎症反应中也可能扮演重要角色，但是以上推论并没有直接证据支持。本实验选用人骨骼肌细胞作为对象，研究TLR4的表达水平对炎症因子活性和胰岛素抵抗的影响，以及脂肪酸对TLR4活性及其下游炎症因子的影响。研究方法： 1.棕榈酸和DHA(100μmol/L、200μmol/L、400μmol/L)干预人骨骼肌细胞4h，8h，12h，24h后，用MTT法测定其对骨骼肌细胞增殖的影响。棕榈酸和DHA干预人骨骼肌细胞24h后，用葡萄糖氧化酶法（GOD-POD法）测定细胞培养液中葡萄糖含量；用Western Blot法检测TLR4，MyD88，，CRP的表达；Real-time PCR法检测TLR4，MyD88，CRP，TNF-α，IL-6mRNA的表达。 2.用LPS和PMB分别作用人骨骼肌细胞24h，用Western Blot法检测TLR4，MyD88的表达；Real-time PCR法检测TLR4，MyD88，CRP，TNF-α，IL-6mRNA的表达；用葡萄糖氧化酶法（GOD-POD法）测定细胞培养液中的葡萄糖含量。研究结果： 1.棕榈酸作用于人骨骼肌细胞，与对照组比较，其葡萄糖摄取能力明显下降（P0.05），TLR4及其下游炎症因子TNF-α，IL-6，CRP表达均明显增高（P0.05）;但TLR4下游适配分子MyD88mRNA显著增高（P0.05），但蛋白水平没有明显差异(P0.05）。相反，DHA作用的人骨骼肌细胞均没有明显变化。 2. TLR4高表达组的炎症因子TNF-α，IL-6，CRP及MyD88的表达明显高于对照组（P0.05），其葡萄糖摄取能力明显下降（P0.05）；TLR4低表达组的炎症因子TNF-α，IL-6，CRP及MyD88的表达明显低于对照组（P0.05）。，其葡萄糖摄取能力没有显著变化（P0.05）。结论：饱和脂肪酸可以诱导人骨骼肌细胞炎症因子的释放，引起葡萄糖摄取能力降低，胰岛素敏感性下降，继而导致胰岛素抵抗。这种作用是通过TLR4介导的。而ω-3多不饱和脂肪酸没有此作用。
[Abstract]:Objectives of the study: Metabolic disease is a worldwide problem that seriously affects people's quality of life. Metabolic diseases are associated with obesity caused by long-term positive energy intake. An important link in obesity leading to other metabolic diseases is the formation of systemic insulin resistance. Therefore, insulin resistance is the key to the formation of metabolic diseases. The main inducing factor of insulin resistance in adipose tissue was the increase of free fatty acids, and the increase of free fatty acids was related to obesity caused by long-term positive energy balance. Obesity is essentially a low-grade inflammatory response. However, the mechanism of inflammatory response caused by obesity is unclear. The body's immune system is carefully regulated, and the inflammatory response process often has one or more key regulatory points. It has been shown that TLR4 may be a key regulatory point in the low-grade inflammation induced by obesity. TLR4 may play an important role in the initiation and transcription of inflammatory factor genes, and may also play an important role in the inflammatory response induced by obesity. But the above reasoning is not supported by direct evidence. In this study, human skeletal muscle cells were selected to study the effects of TLR4 expression level on the activity of inflammatory factors and insulin resistance, and the effects of fatty acids on the activity of TLR4 and its downstream inflammatory factors. Research methods: 1. The effects of palmitic acid and DHA(100 渭 mol / L ~ (200 渭 mol 路L ~ (-1) on the proliferation of human skeletal muscle cells were measured by MTT assay. After intervention with palmitic acid and DHA for 24 hours, the glucose content in culture medium was determined by glucose oxidase assay (GOD-POD), and the expression of TLR4, MyD88hCRP was detected by Western Blot method. Real-time PCR method was used to detect the mRNA expression of TLR4MID88CRP- 伪 TNF- 伪 in human skeletal muscle cells. 2. Human skeletal muscle cells were incubated with LPS and PMB for 24 h respectively. The expression of TLR4- MyD88 was detected by Western Blot method and the expression of TNF- 伪 -IL-6 mRNA was detected by real-time PCR method. The glucose content in cell culture medium was determined by glucose oxidase method (GOD-POD). Results of the study: 1. Compared with the control group, the glucose uptake capacity of palmitic acid on human skeletal muscle cells was significantly decreased. The expression of TLR4 and its down-stream inflammatory factor, TNF- 伪 / TNF- 伪, was significantly increased in human skeletal muscle cells, but the expression of MyD88mRNA, an adaptation molecule in the downstream of TLR4, was significantly higher than that in the control group, but there was no significant difference in protein level between them. On the contrary, there was no significant change in human skeletal muscle cells treated with DHA. 2. The expression of TNF- 伪 IL-6CRP and MyD88 in the high expression group of TLR4 was significantly higher than that in the control group (P 0.05). The glucose uptake capacity of the high expression group was significantly lower than that of the control group. The expression of the inflammatory factor TNF- 伪, IL-6, TLR4 and MyD88 in the low expression group was significantly lower than that in the control group. Conclusion: Saturated fatty acids can induce the release of inflammatory cytokines from human skeletal muscle cells, resulting in a decrease in glucose uptake, insulin sensitivity and insulin resistance. This effect is mediated by TLR4. Omega-3 polyunsaturated fatty acids had no such effect.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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