Snail在腎小管上皮間質(zhì)轉(zhuǎn)化過程中的表達及意義
發(fā)布時間：2019-02-23 22:37
【摘要】：目的:從組織和細(xì)胞水平上探討Snail的表達與腎小管上皮間質(zhì)轉(zhuǎn)化(EMT)及腎間質(zhì)纖維化(TIF)中的關(guān)系;觀察轉(zhuǎn)染Snail基因后人腎小管上皮細(xì)胞(HK-2)miRNA表達譜的變化。方法:(1)采用HE、PAS、PASM、Masson染色評估40例IgA腎病患者腎穿組織腎小管間質(zhì)病理變化,根據(jù)腎小管萎縮及間質(zhì)纖維化程度分為輕度、中度、重度三組,選取4例正常腎臟組織作為對照,采用免疫組化方法檢測Snail及EMT相關(guān)蛋白Vimentin、SMA、E-cadherin的表達。(2)構(gòu)建過表達Snail的質(zhì)粒,采用陽性脂質(zhì)體LipofectamineTM2000轉(zhuǎn)染HK-2細(xì)胞,24 h后倒置顯微鏡下觀察細(xì)胞形態(tài)變化。(3)將體外培養(yǎng)的HK-2細(xì)胞分為正常對照組、空轉(zhuǎn)染組、Snail基因轉(zhuǎn)染組。實時熒光定量(qPCR)檢測Snail基因的表達,RT-PCR及Western blot方法檢測Snail、Vimentin、SMA、E-cadherin蛋白和基因在HK-2細(xì)胞中的表達。進一步借助基因芯片篩選出差異表達的miRNAs。結(jié)果:(1)免疫組化結(jié)果顯示,Snail、Vimentin及SMA在IgA腎病組織中高表達,E-cadherin在IgA腎病組織中低表達;IgA腎病組織中Snail與Vimentin及SMA蛋白的表達呈正相關(guān),與E-cadherin蛋白的表達呈負(fù)相關(guān);且TIF程度越高,Snail蛋白表達越高。(2)經(jīng)測序驗證本實驗成功構(gòu)建出過表達Snail的質(zhì)粒,轉(zhuǎn)染24 h后借助倒置顯微鏡觀察,空白對照組及空轉(zhuǎn)染組HK-2細(xì)胞形態(tài)呈鋪路石樣,Snail基因轉(zhuǎn)染組中HK-2細(xì)胞形態(tài)呈紡錘形,間隙增寬,兩者形態(tài)上差別明顯。(3)qPCR結(jié)果顯示,Snail轉(zhuǎn)染組中Snail mRNA的相對表達量明顯高于空白對照組及空轉(zhuǎn)染組,差異有統(tǒng)計學(xué)意義(P㩳0.01)。RT-PCR及Western blot檢測結(jié)果顯示,與對照組相比,Snail轉(zhuǎn)染組Snail、Vimentin、SMA在基因和蛋白水平表達均升高,E-cadherin蛋白表達降低,差異具有統(tǒng)計學(xué)意義(P㩳0.05)�；蛐酒Y(jié)果表明,Snail轉(zhuǎn)染HK-2細(xì)胞后,篩選出5個明顯差異表達的miRNA,預(yù)測出5026個可能的潛在靶基因。結(jié)論:Snail表達與腎小管上皮間質(zhì)轉(zhuǎn)化及腎間質(zhì)纖維化關(guān)系密切;差異表達的miRNAs是否參與了Snail促進EMT及TIF過程的發(fā)生發(fā)展有待進一步研究。
[Abstract]:Aim: to investigate the relationship between the expression of Snail and tubuloepithelial interstitial transformation (EMT) and interstitial fibrosis (TIF) in renal tubule epithelial cells (HK-2), and to observe the changes of miRNA expression profile in human renal tubular epithelial cells (HK-2) after transfection of Snail gene. Methods: (1) HE,PAS,PASM,Masson staining was used to evaluate the pathological changes of renal tubulointerstitial tissue in 40 patients with IgA nephropathy. According to the degree of tubular atrophy and interstitial fibrosis, they were divided into three groups: mild, moderate and severe. The expression of Snail and EMT related protein Vimentin,SMA,E-cadherin was detected by immunohistochemical method in 4 normal kidney tissues. (2) the expression plasmid of Snail was constructed, and the HK-2 cells were transfected with positive liposome LipofectamineTM2000. (3) HK-2 cells cultured in vitro were divided into normal control group, empty transfection group and Snail gene transfection group. The expression of Snail gene was detected by real-time fluorescence quantitative (qPCR), and the expression of Snail,Vimentin,SMA,E-cadherin protein and gene in HK-2 cells was detected by RT-PCR and Western blot methods. Further screening of differentially expressed miRNAs. by gene chip Results: (1) Immunohistochemical results showed that Snail,Vimentin and SMA were highly expressed in IgA nephropathy and E-cadherin was low in IgA nephropathy. The expression of Snail was positively correlated with the expression of Vimentin and SMA protein in IgA nephropathy and negatively correlated with the expression of E-cadherin protein. The higher the degree of TIF was, the higher the expression of Snail protein was. (2) the plasmid expressing Snail was successfully constructed by sequencing. After 24 h of transfection, the morphology of HK-2 cells in blank control group and empty transfection group was like paving stone. The morphology of HK-2 cells in Snail gene transfection group was spindle-shaped and the gap widened. (3) the relative expression of Snail mRNA in Snail transfection group was significantly higher than that in blank control group and empty transfection group. The results of RT-PCR and Western blot analysis showed that compared with the control group, the expression of Snail,Vimentin,SMA and E-cadherin protein in Snail transfection group was higher than that in control group, and the expression of E-cadherin protein was decreased in Snail transfection group. The difference was statistically significant (P0. 05). The results of gene chip analysis showed that after Snail transfection into HK-2 cells, five differentially expressed miRNA, genes were screened and 5026 potential target genes were predicted. Conclusion: the expression of Snail is closely related to tubule epithelial interstitial transformation and renal interstitial fibrosis, and whether the differentially expressed miRNAs is involved in the process of EMT and TIF promotion by Snail needs further study.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692
本文編號：2429284
[Abstract]:Aim: to investigate the relationship between the expression of Snail and tubuloepithelial interstitial transformation (EMT) and interstitial fibrosis (TIF) in renal tubule epithelial cells (HK-2), and to observe the changes of miRNA expression profile in human renal tubular epithelial cells (HK-2) after transfection of Snail gene. Methods: (1) HE,PAS,PASM,Masson staining was used to evaluate the pathological changes of renal tubulointerstitial tissue in 40 patients with IgA nephropathy. According to the degree of tubular atrophy and interstitial fibrosis, they were divided into three groups: mild, moderate and severe. The expression of Snail and EMT related protein Vimentin,SMA,E-cadherin was detected by immunohistochemical method in 4 normal kidney tissues. (2) the expression plasmid of Snail was constructed, and the HK-2 cells were transfected with positive liposome LipofectamineTM2000. (3) HK-2 cells cultured in vitro were divided into normal control group, empty transfection group and Snail gene transfection group. The expression of Snail gene was detected by real-time fluorescence quantitative (qPCR), and the expression of Snail,Vimentin,SMA,E-cadherin protein and gene in HK-2 cells was detected by RT-PCR and Western blot methods. Further screening of differentially expressed miRNAs. by gene chip Results: (1) Immunohistochemical results showed that Snail,Vimentin and SMA were highly expressed in IgA nephropathy and E-cadherin was low in IgA nephropathy. The expression of Snail was positively correlated with the expression of Vimentin and SMA protein in IgA nephropathy and negatively correlated with the expression of E-cadherin protein. The higher the degree of TIF was, the higher the expression of Snail protein was. (2) the plasmid expressing Snail was successfully constructed by sequencing. After 24 h of transfection, the morphology of HK-2 cells in blank control group and empty transfection group was like paving stone. The morphology of HK-2 cells in Snail gene transfection group was spindle-shaped and the gap widened. (3) the relative expression of Snail mRNA in Snail transfection group was significantly higher than that in blank control group and empty transfection group. The results of RT-PCR and Western blot analysis showed that compared with the control group, the expression of Snail,Vimentin,SMA and E-cadherin protein in Snail transfection group was higher than that in control group, and the expression of E-cadherin protein was decreased in Snail transfection group. The difference was statistically significant (P0. 05). The results of gene chip analysis showed that after Snail transfection into HK-2 cells, five differentially expressed miRNA, genes were screened and 5026 potential target genes were predicted. Conclusion: the expression of Snail is closely related to tubule epithelial interstitial transformation and renal interstitial fibrosis, and whether the differentially expressed miRNAs is involved in the process of EMT and TIF promotion by Snail needs further study.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)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 王冬;李永君;丁楠;王均云;楊瓊;楊雅冉;李艷明;方向東;趙華;;miRNA調(diào)控惡性黑色素瘤細(xì)胞上皮-間充質(zhì)轉(zhuǎn)化的分子網(wǎng)絡(luò)及機制[J];遺傳;2015年07期
2 Minal Garg;;Epithelial-mesenchymal transition-activating transcription factors-multifunctional regulators in cancer[J];World Journal of Stem Cells;2013年04期
3 鄭月娥;李里香;;EMT誘導(dǎo)因子的最新研究進展[J];臨床與實驗病理學(xué)雜志;2013年03期
4 尤小寒;章慧娣;蘇震;薛向陽;黃朝興;;與大鼠腎間質(zhì)纖維化相關(guān)microRNA的初步研究[J];中華腎臟病雜志;2012年10期
5 徐沙沙;項鋒鋼;黨受琴;冉雯雯;李宏;;NF-kB與Slug在非小細(xì)胞肺癌及其上皮間質(zhì)轉(zhuǎn)化中的作用(英文)[J];現(xiàn)代生物醫(yī)學(xué)進展;2012年17期
6 查運紅;梅元武;毛玲;賀杰峰;殷濤;張立;王全勝;;Snail基因修飾對骨髓基質(zhì)干細(xì)胞骨架結(jié)構(gòu)穩(wěn)定作用及促遷移和對無血清培養(yǎng)誘導(dǎo)細(xì)胞凋亡的保護作用研究[J];生物工程學(xué)報;2007年04期
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