FoxO3a表达下调对人肾小管上皮细胞EMT的影响

发布时间：2018-04-13 21:03

本文选题：肾小管上皮细胞 + FoxO3a　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:1.检测Fox O3a转录因子是否与人肾小管上皮细胞上皮-间质转化(EMT)过程有关;2.探讨在人肾小管上皮细胞中Fox O3a是否通过调节β-Catenin对细胞EMT产生影响。方法:1.10 ng/ml的TGF-β1处理HK-2细胞后采用蛋白免疫印迹技术(Western blotting)检测检测E-cadherin、Vimentin、α-SMA表达量变化;2.利用免疫荧光染色法检测Vimentin、α-SMA的表达量变化,以及药物处理后细胞的形态变化;3.应用蛋白免疫印迹技术(Western blotting)分析EMT细胞中Fox O3a、p-Fox O3a蛋白表达量的变化;4.使用激光共聚焦显微镜检测TGF-β1诱导HK-2细胞后,Fox O3a在细胞中的亚分布变化;5.将Fox O3a-si RNA转入HK-2细胞后,通过采用蛋白免疫印迹技术(Western blotting)和RT-PCR检测Fox O3a是否敲低成功,Western blotting检测E-cadherin、α-SMA表达量变化,验证Fox O3a对细胞EMT程度的影响;6.应用Western blot与免疫荧光染色法检测EMT细胞与Fox O3a敲低细胞中β-Catenin的表达变化。结果:1.TGF-β1处理HK-2细胞后,Western blotting检测显示E-Cadherin表达显著降低(P0.01),Vimentin、α-SMA表达量增加(P0.05);免疫荧光染色实验表明Vimentin、α-SMA蛋白的荧光强度显著增强,细胞由上皮细胞呈现的“铺路石”椭圆结构转变为长梭形纤维细胞形态,证明EMT细胞模型建立成功。2.蛋白免疫印迹实验表明发生EMT的HK-2细胞中,Fox O3a表达量降低(P0.01),而p-Fox O3a则显著升高(P0.05),表明Fox O3a与肾小管细胞EMT过程相关。3.激光共聚焦结果显示与正常HK-2细胞相比,EMT细胞中胞核Fox O3a含量减少,而胞质中Fox O3a表达量显著。4.Western blotting和RT-PCR结果分析得出转染Fox O3a-si RNA后,细胞中的Fox O3a表达量显著降低(P0.01),且E-Cadherin表达显著降低(P0.01),α-SMA表达量增加(P0.05),细胞出现EMT现象。表明Fox O3a含量降低,能促使人肾小管上皮细胞发生EMT。5.Western blotting和免疫荧光实验显示EMT细胞与Fox O3a敲低的细胞中β-Catenin的表达都显著升高,说明降低转录因子Fox O3a的表达,可以促使β-Catenin蛋白的表达量增加,具体机制有待于进一步研究。结论:1.转录因子Fox O3a与HK-2细胞的EMT过程有关。并且TGF-β1诱导Fox O3a转位出核,抑制FOXO3a的表达可以使HK-2细胞发生EMT。2.Fox O3a的表达抑制后,可能通过上调β-Catenin蛋白的表达量,从而促进肾小管上皮细胞的上皮-间质转化。
[Abstract]:Purpose 1.Whether Fox O3a transcription factor is related to the epithelial-interstitial transformation of human renal tubular epithelial cells.To investigate the effect of Fox O 3a on EMT by regulating 尾 -Catenin in human renal tubular epithelial cells.Methods after HK-2 cells were treated with TGF- 尾 1 of 1. 10 ng/ml, the expression of E-cadherin and 伪 -SMA were detected by Western blotting.The expression of Vimentin, 伪 -SMA and the morphological changes of Vimentin, 伪 -SMA were detected by immunofluorescence staining.Western blotting technique was used to analyze the expression of Fox O3aP- Fox O3a protein in EMT cells.The subdistribution of HK-2 cells induced by TGF- 尾 1 was detected by confocal laser microscopy.After Fox O3a-si RNA was transferred into HK-2 cells, the expression of E-cadherin and 伪 -SMA was detected by Western blotting and RT-PCR. The effect of Fox O 3a on EMT degree was verified.The expression of 尾 -Catenin in EMT and Fox O 3a knockout cells was detected by Western blot and immunofluorescence staining.Results 1. HK-2 cells treated with TGF- 尾 1 significantly decreased the expression of E-Cadherin and increased the expression of 伪 -SMA, and the fluorescence intensity of Vimentin, 伪 -SMA protein was significantly increased by immunofluorescence staining.The cells changed from "paving stone" elliptical structure of epithelial cells to long fusiform fibrous cells, which proved that EMT cell model was established successfully. 2.Western blot analysis showed that the expression of Fox O3a in HK-2 cells with EMT decreased P0.01a, while p-Fox O3a increased P0.05a, suggesting that Fox O3a was associated with the EMT process of renal tubule cells.The results of laser confocal focus showed that the content of nuclear Fox O3a in EMT cells was lower than that in normal HK-2 cells, while the expression of Fox O3a in cytoplasm was significant. 4. The results of Western blotting and RT-PCR showed that after transfection of Fox O3a-si RNA, the expression of Fox O 3a was significantly higher than that of normal cells.The expression of Fox O3a decreased significantly, and the expression of E-Cadherin decreased significantly. The expression of 伪 -SMA increased the expression of P0.05A, and EMT appeared in the cells.The results showed that the decrease of Fox O 3a content induced the occurrence of EMT.5.Western blotting in human renal tubular epithelial cells and the expression of 尾 -Catenin in EMT cells and Fox O 3a knockdown cells were significantly increased by immunofluorescence assay, which indicated that the expression of Fox O 3a was decreased, and the expression of 尾 -Catenin was significantly increased in both EMT cells and Fox O 3a knockout cells.The expression of 尾-Catenin protein was increased, and the specific mechanism needed to be further studied.Conclusion 1.Transcription factor Fox O 3a is related to the EMT process of HK-2 cells.TGF- 尾 1 induced the translocation of Fox O 3a into the extranuclear cells, which inhibited the expression of EMT.2.Fox O 3a in HK-2 cells, which may promote the epithelial-interstitial transformation of renal tubular epithelial cells by up-regulating the expression of 尾 -Catenin protein.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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