本文選題：AQP9 + FoxO1��； 參考：《重慶醫(yī)科大學》2011年碩士論文

【摘要】：目的:觀察FoxO1基因沉默對水通道蛋白9(AQP9)在正常人肝細胞L-02中表達的影響。 方法:將4條特異性干擾質(zhì)粒FoxO1-shRNA1~4經(jīng)脂質(zhì)體法瞬時轉(zhuǎn)染L-02細胞,熒光顯微鏡下觀察轉(zhuǎn)染效率,篩選沉默效率最佳的一條質(zhì)粒載體轉(zhuǎn)染肝細胞,經(jīng)RT-PCR法檢測其對AQP9基因表達的影響;細胞免疫熒光、Western blot法檢測其對AQP9蛋白表達的影響;并在轉(zhuǎn)染后不同時間點收集細胞,實時熒光定量PCR檢測轉(zhuǎn)染后FOXO1抑制AQP9轉(zhuǎn)錄表達的時間譜。 結(jié)果:4條特異性干擾質(zhì)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,與對照組相比,AQP9在L-02細胞中的mRNA及蛋白水平的表達水平下降差異有統(tǒng)計學意義(P0.05);定量PCR檢測顯示,AQP9的表達隨轉(zhuǎn)染時間的延長而降低,在轉(zhuǎn)染后60 h達抑制峰值,差異有統(tǒng)計學意義(P0.05)。 結(jié)論:FoxO1基因沉默對AQP9在正常肝細胞中的表達有確切的影響,AQP9基因和蛋白的表達會隨FoxO1表達降低而降低,在60h達到抑制峰值;本研究為我們進一步采用染色質(zhì)免疫共沉淀技術(shù)探討不同時間點FoxO1與AQP9啟動子區(qū)IRE結(jié)合水平的動態(tài)變化提供了前期實驗依據(jù)。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前1條

1 趙豐瑩;梅浙川;;水通道蛋白9在培養(yǎng)肝細胞脂肪變性模型中的表達及意義[J];重慶醫(yī)科大學學報;2008年08期

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