本文選題：脂肪細(xì)胞 + toll樣受體4�。� 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:toll樣受體4(toll-like receptor 4,TLR4)在機(jī)體天然免疫系統(tǒng)識別病原體過程中發(fā)揮著非常重要的作用。近年來炎癥在糖尿病中的作用受到重視,而AKT信號通路之一就是胰島素信號通路。本實(shí)驗(yàn)旨在研究,在脂肪細(xì)胞中激活TLR4后,對于脂肪細(xì)胞中AKT信號通路有無影響:并且探討天津地區(qū)人群中TLR4基因Asp299Gly、Thr399Ile多態(tài)性以及過氧化物酶體增殖物激活受體γ(peroxisome proliferator-activated receptorγ,PPARγ)基因Prol2Ala多態(tài)性與2型糖尿病(type 2 diabetes mellitus,T2DM)的關(guān)系。 方法:1.用DMEM培養(yǎng)基培養(yǎng)3T3-L1細(xì)胞,待其接觸抑制以后,加入誘導(dǎo)劑使其分化為脂肪細(xì)胞。 2.利用油紅染色的方法對脂肪細(xì)胞進(jìn)行鑒定。 3.在脂肪細(xì)胞中加入脂多糖(lipopolysaccharides,LPS),作用1 h后,提取細(xì)胞總RNA,采用realtime-PCR的方法測定TLR4的mRNA表達(dá)豐度。 4.提取細(xì)胞總蛋白,western blot的方法檢測p-AKT蛋白表達(dá)水平。 5.分別提取365名2型糖尿病人和95名健康人的基因組DNA,PCR擴(kuò)增目的基因片斷。 6.用變性高效液相色譜(denaturing high performance liquidchromatography,DHPLC)篩查TLR4基因Asp299Gly、Thr399Ile位點(diǎn)以及PPARγ基因Prol2Ala位點(diǎn)多態(tài)性。 結(jié)果:1.誘導(dǎo)3T3-L1細(xì)胞,誘導(dǎo)分化率比較高。誘導(dǎo)7-8天,90%以上的細(xì)胞分化成為脂肪細(xì)胞。 2.油紅染色結(jié)果呈陽性,大部分脂肪細(xì)胞染成紅色,胞漿中見大量脂滴。 3.脂肪細(xì)胞在用LPS作用1 h后,TLR4的mRNA的表達(dá)明顯增加(P＜0.05)。 4.在脂肪細(xì)胞中加入LPS后,p-AKT蛋白表達(dá)水平下降(P＜0.05)。 5.在T2DM組和對照組均未篩查到TLR4基因Asp299Gly、Thr399Ile突變基因型的存在。 6.T2DM組和健康人對照組PPARγ基因Prol2Ala位點(diǎn)P/A的基因型頻率分別為0.074和0.032(27/365和3/95),兩組相比差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 結(jié)論:1.誘導(dǎo)3T3-L1細(xì)胞分化成脂肪細(xì)胞,具有較高的分化率,可達(dá)90%以上。 2.脂肪細(xì)胞中加入LPS后,TLR4的mRNA的表達(dá)豐度增加,而p-AKT水平下降,初步說明TLR4的表達(dá)可以抑制脂肪細(xì)胞中AKT信號通路。 3.天津地區(qū)人群未篩查到TLR4基因Asp299Gly、Thr399Ile突變基因型,其多態(tài)性與T2DM無明顯關(guān)聯(lián)。 4.天津地區(qū)人群PPARγ基因Prol2Ala多態(tài)性與T2DM無明顯關(guān)聯(lián)。 5.DHPLC是一種快速有效的基因突變篩查方法,可以用來篩查2型糖尿病易感基因,預(yù)測糖尿病發(fā)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 緯 PPAR- 緯 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 緯 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 緯 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 緯 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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341;R587.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 高從容,鄒大進(jìn),梅小斌,郭品娥,張樂枝;2型糖尿病患者血清高敏感C反應(yīng)蛋白水平與大血管病變的關(guān)系及辛伐他汀的干預(yù)作用[J];上海醫(yī)學(xué);2004年01期

2 李嘉強(qiáng);炎癥標(biāo)志物與冠心病[J];心血管病學(xué)進(jìn)展;2003年03期

，

本文編號：1955967