FoxO1-shRNA干扰质粒对水通道蛋白9在正常人肝细胞中表达的影响

发布时间：2018-04-20 18:26

本文选题：AQP9 + FoxO1　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的:观察FoxO1基因沉默对水通道蛋白9(AQP9)在正常人肝细胞L-02中表达的影响。方法:将4条特异性干扰质粒FoxO1-shRNA1~4经脂质体法瞬时转染L-02细胞,荧光显微镜下观察转染效率,筛选沉默效率最佳的一条质粒载体转染肝细胞,经RT-PCR法检测其对AQP9基因表达的影响;细胞免疫荧光、Western blot法检测其对AQP9蛋白表达的影响;并在转染后不同时间点收集细胞,实时荧光定量PCR检测转染后FOXO1抑制AQP9转录表达的时间谱。结果:4条特异性干扰质粒FoxO1-shRNA1~4均有不同程度的沉默效果,其中以FoxO1-shRNA2沉默效果最佳,与对照组相比,AQP9在L-02细胞中的mRNA及蛋白水平的表达水平下降差异有统计学意义(P0.05);定量PCR检测显示,AQP9的表达随转染时间的延长而降低,在转染后60 h达抑制峰值,差异有统计学意义(P0.05)。结论:FoxO1基因沉默对AQP9在正常肝细胞中的表达有确切的影响,AQP9基因和蛋白的表达会随FoxO1表达降低而降低,在60h达到抑制峰值;本研究为我们进一步采用染色质免疫共沉淀技术探讨不同时间点FoxO1与AQP9启动子区IRE结合水平的动态变化提供了前期实验依据。
[Abstract]:Aim: to investigate the effect of FoxO1 gene silencing on the expression of aquaporin 9 AQP9 in normal human hepatocytes L-02. Methods: four specific interference plasmids (FoxO1-shRNA1~4) were transiently transfected into L-02 cells by liposome method. The transfection efficiency was observed under fluorescence microscope. The best plasmid vector was selected for transfection into hepatocytes. The effect of AQP9 gene expression was detected by RT-PCR method. The effect of FOXO1 on the expression of AQP9 protein was detected by cell immunofluorescence assay, and the time spectrum of FOXO1 inhibiting the expression of AQP9 transcription was detected by real-time fluorescence quantitative PCR at different time points after transfection. Results all of the four specific interference plasmids FoxO1-shRNA1~4 showed different silencing effects, among which FoxO1-shRNA2 was the best. Compared with the control group, the expression of mRNA and protein in L-02 cells was significantly lower than that in the control group, and the quantitative PCR assay showed that the expression of pAQP9 decreased with the extension of transfection time, and reached the inhibitory peak at 60 h after transfection. The difference was statistically significant (P 0.05). Conclusion the expression of AQP9 gene and protein decreased with the decrease of FoxO1 expression, and reached the inhibition peak at 60 h after the silencing of the cell line: 1 FoxO1 gene silencing had a definite effect on the expression of AQP9 in normal hepatocytes. This study provides a preliminary experimental basis for the further use of chromatin immunoprecipitation technique to study the dynamic changes of IRE binding level between FoxO1 and AQP9 promoter at different time points.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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