【摘要】：目的:從組織和細(xì)胞水平上探討Snail的表達(dá)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(dá)譜的變化。方法:(1)采用HE、PAS、PASM、Masson染色評(píng)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(duì)照,采用免疫組化方法檢測(cè)Snail及EMT相關(guān)蛋白Vimentin、SMA、E-cadherin的表達(dá)。(2)構(gòu)建過(guò)表達(dá)Snail的質(zhì)粒,采用陽(yáng)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(duì)照組、空轉(zhuǎn)染組、Snail基因轉(zhuǎn)染組。實(shí)時(shí)熒光定量(qPCR)檢測(cè)Snail基因的表達(dá),RT-PCR及Western blot方法檢測(cè)Snail、Vimentin、SMA、E-cadherin蛋白和基因在HK-2細(xì)胞中的表達(dá)。進(jìn)一步借助基因芯片篩選出差異表達(dá)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(dá),E-cadherin在IgA腎病組織中低表達(dá);IgA腎病組織中Snail與Vimentin及SMA蛋白的表達(dá)呈正相關(guān),與E-cadherin蛋白的表達(dá)呈負(fù)相關(guān);且TIF程度越高,Snail蛋白表達(dá)越高。(2)經(jīng)測(cè)序驗(yàn)證本實(shí)驗(yàn)成功構(gòu)建出過(guò)表達(dá)Snail的質(zhì)粒,轉(zhuǎn)染24 h后借助倒置顯微鏡觀察,空白對(duì)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(duì)表達(dá)量明顯高于空白對(duì)照組及空轉(zhuǎn)染組,差異有統(tǒng)計(jì)學(xué)意義(P㩳0.01)。RT-PCR及Western blot檢測(cè)結(jié)果顯示,與對(duì)照組相比,Snail轉(zhuǎn)染組Snail、Vimentin、SMA在基因和蛋白水平表達(dá)均升高,E-cadherin蛋白表達(dá)降低,差異具有統(tǒng)計(jì)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(gè)明顯差異表達(dá)的miRNA,預(yù)測(cè)出5026個(gè)可能的潛在靶基因。結(jié)論:Snail表達(dá)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(dá)的miRNAs是否參與了Snail促進(jìn)EMT及TIF過(guò)程的發(fā)生發(fā)展有待進(jìn)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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R692

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