本文選題：腎小管上皮細胞 + FoxO3a��； 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:1.檢測Fox O3a轉(zhuǎn)錄因子是否與人腎小管上皮細胞上皮-間質(zhì)轉(zhuǎn)化(EMT)過程有關(guān);2.探討在人腎小管上皮細胞中Fox O3a是否通過調(diào)節(jié)β-Catenin對細胞EMT產(chǎn)生影響。方法:1.10 ng/ml的TGF-β1處理HK-2細胞后采用蛋白免疫印跡技術(shù)(Western blotting)檢測檢測E-cadherin、Vimentin、α-SMA表達量變化;2.利用免疫熒光染色法檢測Vimentin、α-SMA的表達量變化,以及藥物處理后細胞的形態(tài)變化;3.應用蛋白免疫印跡技術(shù)(Western blotting)分析EMT細胞中Fox O3a、p-Fox O3a蛋白表達量的變化;4.使用激光共聚焦顯微鏡檢測TGF-β1誘導HK-2細胞后,Fox O3a在細胞中的亞分布變化;5.將Fox O3a-si RNA轉(zhuǎn)入HK-2細胞后,通過采用蛋白免疫印跡技術(shù)(Western blotting)和RT-PCR檢測Fox O3a是否敲低成功,Western blotting檢測E-cadherin、α-SMA表達量變化,驗證Fox O3a對細胞EMT程度的影響;6.應用Western blot與免疫熒光染色法檢測EMT細胞與Fox O3a敲低細胞中β-Catenin的表達變化。結(jié)果:1.TGF-β1處理HK-2細胞后,Western blotting檢測顯示E-Cadherin表達顯著降低(P0.01),Vimentin、α-SMA表達量增加(P0.05);免疫熒光染色實驗表明Vimentin、α-SMA蛋白的熒光強度顯著增強,細胞由上皮細胞呈現(xiàn)的“鋪路石”橢圓結(jié)構(gòu)轉(zhuǎn)變?yōu)殚L梭形纖維細胞形態(tài),證明EMT細胞模型建立成功。2.蛋白免疫印跡實驗表明發(fā)生EMT的HK-2細胞中,Fox O3a表達量降低(P0.01),而p-Fox O3a則顯著升高(P0.05),表明Fox O3a與腎小管細胞EMT過程相關(guān)。3.激光共聚焦結(jié)果顯示與正常HK-2細胞相比,EMT細胞中胞核Fox O3a含量減少,而胞質(zhì)中Fox O3a表達量顯著。4.Western blotting和RT-PCR結(jié)果分析得出轉(zhuǎn)染Fox O3a-si RNA后,細胞中的Fox O3a表達量顯著降低(P0.01),且E-Cadherin表達顯著降低(P0.01),α-SMA表達量增加(P0.05),細胞出現(xiàn)EMT現(xiàn)象。表明Fox O3a含量降低,能促使人腎小管上皮細胞發(fā)生EMT。5.Western blotting和免疫熒光實驗顯示EMT細胞與Fox O3a敲低的細胞中β-Catenin的表達都顯著升高,說明降低轉(zhuǎn)錄因子Fox O3a的表達,可以促使β-Catenin蛋白的表達量增加,具體機制有待于進一步研究。結(jié)論:1.轉(zhuǎn)錄因子Fox O3a與HK-2細胞的EMT過程有關(guān)。并且TGF-β1誘導Fox O3a轉(zhuǎn)位出核,抑制FOXO3a的表達可以使HK-2細胞發(fā)生EMT。2.Fox O3a的表達抑制后,可能通過上調(diào)β-Catenin蛋白的表達量,從而促進腎小管上皮細胞的上皮-間質(zhì)轉(zhuǎn)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 偽 -SMA were detected by Western blotting.The expression of Vimentin, 偽 -SMA and the morphological changes of Vimentin, 偽 -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 偽 -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 偽 -SMA, and the fluorescence intensity of Vimentin, 偽 -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 偽 -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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692

【參考文獻】

相關(guān)期刊論文 前6條

1 唐娜;肖建華;;TGF-β與轉(zhuǎn)錄因子在肝纖維化中的相互作用[J];微生物學免疫學進展;2016年06期

2 張琳;賀明;;Sirtuins在慢性腎臟疾病發(fā)生、發(fā)展中的作用[J];上海交通大學學報(醫(yī)學版);2015年04期

3 Richard Seonghun Nho;Polla Hergert;;FoxO3a and disease progression[J];World Journal of Biological Chemistry;2014年03期

4 彭婷;杜海堅;李理;李偉峰;黃文杰;;吉非替尼通過增加Foxo3a活性抑制肺纖維化小鼠上皮-間質(zhì)轉(zhuǎn)分化[J];中國病理生理雜志;2014年03期

5 鐘振;劉益飛;劉俊華;;FOXO3a基因在癌癥中的研究進展[J];中國腫瘤外科雜志;2013年03期

6 楊小利;歐周羅;邵志敏;;多功能的轉(zhuǎn)錄因子FOXO3a[J];生命的化學;2012年05期

，

本文編號：1746185