Caveolin-1对3T3-L1细胞生长的影响及其在胰岛素信号通路中的作用

发布时间：2018-01-11 12:11

  本文关键词：Caveolin-1对3T3-L1细胞生长的影响及其在胰岛素信号通路中的作用　出处：《南华大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： Caveolin-1 IR GLUT4

【摘要】：目的：Caveolin-1基因对3T3-L1细胞生长特性影响及其在胰岛素信号通路中的作用机制。 方法：将携带有CAVl全长片段(CAVl)、CAVl沉默的CAVRNAi载体分别转染3T3-L1细胞并通过相应的生物素抗性标记筛选建立稳定细胞株;用Western—Blot检测各组CAVl蛋白的表达，流式细胞术检测细胞所处的周期;用免疫荧光法观测胰岛素刺激前后胰岛素通路中相关蛋白CAVl、GLUT4、IR蛋白的表达情况。用Western—Blot检测CAVl、GLUT4、IR蛋白的定位和表达情况，探讨CAVl在胰岛素信号通路中的作用。 结果：转染pcDNA3．1(-)/NT—GFP—CAVl的3T3一L1细胞其CAVl的表达明显强于其他各组(PO．05)；转染CAVl Lentiviral RNAi组CAVl的表达明显低于其他各组(PO．05)，3T3组与GFP组无明显差别(PO.05) 流式细胞仪检测结果显示，CAVl-GFP组GO/C1期细胞比例明显升高，而G2／M期细胞比例明显降低(PO．05)，RNAi组GO/C1期细胞比例明显降低，而GUM期细胞比例明显升高(PO．05)，而空载体组和正常3T3细胞组之间无统计学差异(PO．05)。 分别用0MU/L、10MU/L及300MU/L RI处理6 h后荧光显微镜下观察Caveolin一1及IR的表达。荧光显微镜下观察：CAVl、3T3、GFP组CAVl及RI呈现红色荧光，RNAi组中CAVl为绿色荧光，散在的分布在胞质中。总的来看，CAVl组荧光强度最强，3T3组及GFP组居中，RNAi组荧光强度最弱。除RNAi组外，其余三组细胞其荧光强度均随RI的浓度升高而增强，在RI浓度为300MU/L时，，荧光强度最强，这种趋势在CAVl组最显著，GFP组及3T3组不明显，而RNAi组无这种趋势。 四个组别的对数生长期细胞分别用0MU/L及300MU/L浓度RI处理6h后，收集蛋白，Wsetern—Blot检测细胞CAVl、IR、GLUT4蛋白的表达。CAVl、3T3、GFP组予以RI 300MU/L处理后CAVl蛋白的表达明显强于无RI处理组(PO．05)，RNAi组此差别不明显(PO．05)。且CAVl蛋白的表达在CAVl组强度最强，3T3组及GFP组居中，RNAi强度最弱(PO．05)。 IR蛋白及GLUT4的表达亦符合上述规律。 结论：1、成功建立了稳定过表达CAVl基因全长的3T3-L1细胞系，成功建立CAVl基因沉默的3T3-L1细胞系。 2、稳定过表达CAVl基因对3T3-L1细胞的生长具有促进作用。 3、胰岛素刺激能够呈浓度依赖性诱导CAVl蛋白、IR蛋白及GLUT4蛋白的表达，且此趋势在过表达CAVl基因的3T3-L1细胞中明显，在CAVl基因沉默组中缺乏。
[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 .

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】

相关期刊论文 前1条

1 徐阳炎,杨慧龄,涂剑,何淑雅,廖端芳;pcDNA3.1/NT-GFP小凹蛋白1及突变体表达载体的构建及功能分析[J];中国动脉硬化杂志;2005年03期

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