3T3-L1细胞TLR4的表达对AKT信号通路的影响

发布时间：2018-05-30 16:30

本文选题：脂肪细胞 + toll样受体4　；参考：《天津医科大学》2008年硕士论文

【摘要】： 目的:toll样受体4(toll-like receptor 4,TLR4)在机体天然免疫系统识别病原体过程中发挥着非常重要的作用。近年来炎症在糖尿病中的作用受到重视,而AKT信号通路之一就是胰岛素信号通路。本实验旨在研究,在脂肪细胞中激活TLR4后,对于脂肪细胞中AKT信号通路有无影响:并且探讨天津地区人群中TLR4基因Asp299Gly、Thr399Ile多态性以及过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)基因Prol2Ala多态性与2型糖尿病(type 2 diabetes mellitus,T2DM)的关系。方法:1.用DMEM培养基培养3T3-L1细胞,待其接触抑制以后,加入诱导剂使其分化为脂肪细胞。 2.利用油红染色的方法对脂肪细胞进行鉴定。 3.在脂肪细胞中加入脂多糖(lipopolysaccharides,LPS),作用1 h后,提取细胞总RNA,采用realtime-PCR的方法测定TLR4的mRNA表达丰度。 4.提取细胞总蛋白,western blot的方法检测p-AKT蛋白表达水平。 5.分别提取365名2型糖尿病人和95名健康人的基因组DNA,PCR扩增目的基因片断。 6.用变性高效液相色谱(denaturing high performance liquidchromatography,DHPLC)筛查TLR4基因Asp299Gly、Thr399Ile位点以及PPARγ基因Prol2Ala位点多态性。结果:1.诱导3T3-L1细胞,诱导分化率比较高。诱导7-8天,90%以上的细胞分化成为脂肪细胞。 2.油红染色结果呈阳性,大部分脂肪细胞染成红色,胞浆中见大量脂滴。 3.脂肪细胞在用LPS作用1 h后,TLR4的mRNA的表达明显增加(P＜0.05)。 4.在脂肪细胞中加入LPS后,p-AKT蛋白表达水平下降(P＜0.05)。 5.在T2DM组和对照组均未筛查到TLR4基因Asp299Gly、Thr399Ile突变基因型的存在。 6.T2DM组和健康人对照组PPARγ基因Prol2Ala位点P/A的基因型频率分别为0.074和0.032(27/365和3/95),两组相比差异无统计学意义(P＞0.05)。结论:1.诱导3T3-L1细胞分化成脂肪细胞,具有较高的分化率,可达90%以上。 2.脂肪细胞中加入LPS后,TLR4的mRNA的表达丰度增加,而p-AKT水平下降,初步说明TLR4的表达可以抑制脂肪细胞中AKT信号通路。 3.天津地区人群未筛查到TLR4基因Asp299Gly、Thr399Ile突变基因型,其多态性与T2DM无明显关联。 4.天津地区人群PPARγ基因Prol2Ala多态性与T2DM无明显关联。 5.DHPLC是一种快速有效的基因突变筛查方法,可以用来筛查2型糖尿病易感基因,预测糖尿病发病的易感性。
[Abstract]:Objective 4(toll-like receptor 4 TLR4) plays an important role in the recognition of pathogens in the innate immune system. In recent years, the role of inflammation in diabetes has been emphasized, and one of the AKT signaling pathways is insulin signaling pathway. The aim of this study was to investigate the activation of TLR4 in adipocytes. To explore the relationship between TLR4 gene Asp299 GlyThr399Ile polymorphism and peroxisome proliferator-activated receptor 纬 PPAR- 纬 gene Prol2Ala polymorphism and type 2 diabetes mellitus type 2 diabetes mellitusu T2DM. Method 1: 1. 3T3-L1 cells were cultured on DMEM medium, and then induced to differentiate into adipocytes after contact inhibition. 2. Fat cells were identified by oil red staining. 3. Lipopolysaccharide (LPSN) was added to adipocytes for 1 hour, then the total RNAs were extracted and the mRNA abundance of TLR4 was determined by realtime-PCR method. 4. The expression of p-AKT protein was detected by western blot extraction. 5. The genomic DNA fragment was amplified by PCR from 365 type 2 diabetic patients and 95 healthy persons. 6. Denaturing high performance was used to screen the polymorphism of TLR4 gene Asp29GlyThr399Ile and PPAR 纬 gene Prol2Ala locus by denaturing high performance liquid chromatography (HPLC). The result is 1: 1. When 3T3-L1 cells were induced, the differentiation rate was higher. More than 90% of the cells were induced to differentiate into adipocytes from 7 to 8 days. 2. Oil red staining showed positive staining, most of the fat cells were stained red, and a large number of lipid droplets were found in the cytoplasm. 3. The expression of TLR4 mRNA in adipocytes was significantly increased after treated with LPS for 1 h (P < 0.05). 4. The expression of p-AKT protein in adipocytes decreased after adding LPS (P < 0.05). 5. No genotypes of Asp299 Glycine Thr399Ile mutation were detected in T2DM and control groups. The genotype frequencies of PPAR 纬 Prol2Ala site P / A in 6.T2DM group and healthy control group were 0.074 and 0.032 ~ 27 / 365 and 3 / 95, respectively. There was no significant difference between the two groups (P > 0.05). Conclusion 1. 3T3-L1 cells were induced to differentiate into adipocytes with a high differentiation rate of more than 90%. 2. The expression of TLR4 mRNA in adipocytes increased and the level of p-AKT decreased after the addition of LPS, which suggested that the expression of TLR4 could inhibit the AKT signaling pathway in adipocytes. 3. The mutation genotype of TLR4 gene Asp299 Glycine Thr399Ile was not screened in Tianjin population, and there was no significant association between the polymorphism and T2DM. 4. There was no significant association between PPAR 纬 gene Prol2Ala polymorphism and T2DM in Tianjin population. 5.DHPLC is a rapid and effective gene mutation screening method, which can be used to screen susceptibility genes of type 2 diabetes mellitus and predict the susceptibility to diabetes.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341;R587.1

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