本文關(guān)鍵詞：Caveolin-1對3T3-L1細(xì)胞生長的影響及其在胰島素信號通路中的作用　出處：《南華大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Caveolin-1 IR GLUT4

【摘要】：目的：Caveolin-1基因?qū)?T3-L1細(xì)胞生長特性影響及其在胰島素信號通路中的作用機(jī)制。 方法：將攜帶有CAVl全長片段(CAVl)、CAVl沉默的CAVRNAi載體分別轉(zhuǎn)染3T3-L1細(xì)胞并通過相應(yīng)的生物素抗性標(biāo)記篩選建立穩(wěn)定細(xì)胞株;用Western—Blot檢測各組CAVl蛋白的表達(dá)，流式細(xì)胞術(shù)檢測細(xì)胞所處的周期;用免疫熒光法觀測胰島素刺激前后胰島素通路中相關(guān)蛋白CAVl、GLUT4、IR蛋白的表達(dá)情況。用Western—Blot檢測CAVl、GLUT4、IR蛋白的定位和表達(dá)情況，探討CAVl在胰島素信號通路中的作用。 結(jié)果：轉(zhuǎn)染pcDNA3．1(-)/NT—GFP—CAVl的3T3一L1細(xì)胞其CAVl的表達(dá)明顯強(qiáng)于其他各組(PO．05)；轉(zhuǎn)染CAVl Lentiviral RNAi組CAVl的表達(dá)明顯低于其他各組(PO．05)，3T3組與GFP組無明顯差別(PO.05) 流式細(xì)胞儀檢測結(jié)果顯示，CAVl-GFP組GO/C1期細(xì)胞比例明顯升高，而G2／M期細(xì)胞比例明顯降低(PO．05)，RNAi組GO/C1期細(xì)胞比例明顯降低，而GUM期細(xì)胞比例明顯升高(PO．05)，而空載體組和正常3T3細(xì)胞組之間無統(tǒng)計(jì)學(xué)差異(PO．05)。 分別用0MU/L、10MU/L及300MU/L RI處理6 h后熒光顯微鏡下觀察Caveolin一1及IR的表達(dá)。熒光顯微鏡下觀察：CAVl、3T3、GFP組CAVl及RI呈現(xiàn)紅色熒光，RNAi組中CAVl為綠色熒光，散在的分布在胞質(zhì)中。總的來看，CAVl組熒光強(qiáng)度最強(qiáng)，3T3組及GFP組居中，RNAi組熒光強(qiáng)度最弱。除RNAi組外，其余三組細(xì)胞其熒光強(qiáng)度均隨RI的濃度升高而增強(qiáng)，在RI濃度為300MU/L時(shí)，，熒光強(qiáng)度最強(qiáng)，這種趨勢在CAVl組最顯著，GFP組及3T3組不明顯，而RNAi組無這種趨勢。 四個(gè)組別的對數(shù)生長期細(xì)胞分別用0MU/L及300MU/L濃度RI處理6h后，收集蛋白，Wsetern—Blot檢測細(xì)胞CAVl、IR、GLUT4蛋白的表達(dá)。CAVl、3T3、GFP組予以RI 300MU/L處理后CAVl蛋白的表達(dá)明顯強(qiáng)于無RI處理組(PO．05)，RNAi組此差別不明顯(PO．05)。且CAVl蛋白的表達(dá)在CAVl組強(qiáng)度最強(qiáng)，3T3組及GFP組居中，RNAi強(qiáng)度最弱(PO．05)。 IR蛋白及GLUT4的表達(dá)亦符合上述規(guī)律。 結(jié)論：1、成功建立了穩(wěn)定過表達(dá)CAVl基因全長的3T3-L1細(xì)胞系，成功建立CAVl基因沉默的3T3-L1細(xì)胞系。 2、穩(wěn)定過表達(dá)CAVl基因?qū)?T3-L1細(xì)胞的生長具有促進(jìn)作用。 3、胰島素刺激能夠呈濃度依賴性誘導(dǎo)CAVl蛋白、IR蛋白及GLUT4蛋白的表達(dá)，且此趨勢在過表達(dá)CAVl基因的3T3-L1細(xì)胞中明顯，在CAVl基因沉默組中缺乏。
[Abstract]:Objective : To investigate the effect of Caveolin - 1 gene on 3T3 - L1 cell growth and its mechanism in insulin signaling pathway . Methods : CAVl ( CAVl ) and CAVl - silenced CAVRNAi vector were transfected into 3T3 - L1 cells respectively . The expression of CAVl , GLUT4 and IR protein was detected by Western - Blot . Western - Blot was used to detect the expression of CAVl , GLUT4 and IR protein . Results : The expression of CAVl of 3T3 - L1 cells transfected with pcDNA3.1 ( - ) / NT - GFP - CAVl was significantly stronger than that in other groups ( PO.05 ) . The results of flow cytometry showed that the percentage of GO / C1 cells increased significantly in CAVl - GFP group , while the percentage of G2 / M cells decreased significantly ( PO.05 ) , and the proportion of GO / C1 cells decreased significantly , while the proportion of cells in GUM phase increased significantly ( PO.05 ) , while there was no statistical difference between the empty vector group and the normal 3T3 cell group ( PO.05 ) . The expression of Caveolin - 1 and IR was observed under fluorescence microscope after 6 h post - treatment with 0MU / L , 10MU / L and 300MU / L RI . The fluorescence intensity of CAVl , 3T3 , GFP was the strongest in the RNAi group . After treated with 0 MU / L and 300 MU / L RI for 6 h , the expression of CAVl , IR , GLUT4 protein was detected by the expression of CAVl , IR and GLUT4 protein in four groups . The expression of CAVl protein was significantly stronger than that in the no RI treatment group ( PO.05 ) , and the difference of the RNAi group was not obvious ( PO.05 ) . The expression of CAVl protein was strongest in CAVl group , and in 3T3 group and GFP group , the intensity of RNAi was weakest ( PO.05 ) . The expression of IR protein and GLUT4 also accords with the above regulation . Conclusion : 1 . 3T3 - L1 cell line stably expressing the full length of CAVl gene has been successfully established , and 3T3 - L1 cell line with CAVl gene silencing has been successfully established . 2 . Stable overexpression of CAVl gene promoted the growth of 3T3 - L1 cells . 3 . Insulin stimulation can induce the expression of CAVl protein , IR protein and GLUT4 protein in concentration - dependent manner , and this trend is obvious in 3T3 - L1 cells expressing CAVl gene , which is absent in CAVl gene silencing group .

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 徐陽炎,楊慧齡,涂劍,何淑雅,廖端芳;pcDNA3.1/NT-GFP小凹蛋白1及突變體表達(dá)載體的構(gòu)建及功能分析[J];中國動(dòng)脈硬化雜志;2005年03期

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