Snail在肾小管上皮间质转化过程中的表达及意义
发布时间：2019-02-23 22:37
【摘要】：目的:从组织和细胞水平上探讨Snail的表达与肾小管上皮间质转化(EMT)及肾间质纤维化(TIF)中的关系;观察转染Snail基因后人肾小管上皮细胞(HK-2)miRNA表达谱的变化。方法:(1)采用HE、PAS、PASM、Masson染色评估40例IgA肾病患者肾穿组织肾小管间质病理变化,根据肾小管萎缩及间质纤维化程度分为轻度、中度、重度三组,选取4例正常肾脏组织作为对照,采用免疫组化方法检测Snail及EMT相关蛋白Vimentin、SMA、E-cadherin的表达。(2)构建过表达Snail的质粒,采用阳性脂质体LipofectamineTM2000转染HK-2细胞,24 h后倒置显微镜下观察细胞形态变化。(3)将体外培养的HK-2细胞分为正常对照组、空转染组、Snail基因转染组。实时荧光定量(qPCR)检测Snail基因的表达,RT-PCR及Western blot方法检测Snail、Vimentin、SMA、E-cadherin蛋白和基因在HK-2细胞中的表达。进一步借助基因芯片筛选出差异表达的miRNAs。结果:(1)免疫组化结果显示,Snail、Vimentin及SMA在IgA肾病组织中高表达,E-cadherin在IgA肾病组织中低表达;IgA肾病组织中Snail与Vimentin及SMA蛋白的表达呈正相关,与E-cadherin蛋白的表达呈负相关;且TIF程度越高,Snail蛋白表达越高。(2)经测序验证本实验成功构建出过表达Snail的质粒,转染24 h后借助倒置显微镜观察,空白对照组及空转染组HK-2细胞形态呈铺路石样,Snail基因转染组中HK-2细胞形态呈纺锤形,间隙增宽,两者形态上差别明显。(3)qPCR结果显示,Snail转染组中Snail mRNA的相对表达量明显高于空白对照组及空转染组,差异有统计学意义(P㩳0.01)。RT-PCR及Western blot检测结果显示,与对照组相比,Snail转染组Snail、Vimentin、SMA在基因和蛋白水平表达均升高,E-cadherin蛋白表达降低,差异具有统计学意义(P㩳0.05)。基因芯片结果表明,Snail转染HK-2细胞后,筛选出5个明显差异表达的miRNA,预测出5026个可能的潜在靶基因。结论:Snail表达与肾小管上皮间质转化及肾间质纤维化关系密切;差异表达的miRNAs是否参与了Snail促进EMT及TIF过程的发生发展有待进一步研究。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
本文编号：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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
【参考文献】
相关期刊论文 前6条
1 王冬;李永君;丁楠;王均云;杨琼;杨雅冉;李艳明;方向东;赵华;;miRNA调控恶性黑色素瘤细胞上皮-间充质转化的分子网络及机制[J];遗传;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 郑月娥;李里香;;EMT诱导因子的最新研究进展[J];临床与实验病理学杂志;2013年03期
4 尤小寒;章慧娣;苏震;薛向阳;黄朝兴;;与大鼠肾间质纤维化相关microRNA的初步研究[J];中华肾脏病杂志;2012年10期
5 徐沙沙;项锋钢;党受琴;冉雯雯;李宏;;NF-kB与Slug在非小细胞肺癌及其上皮间质转化中的作用(英文)[J];现代生物医学进展;2012年17期
6 查运红;梅元武;毛玲;贺杰峰;殷涛;张立;王全胜;;Snail基因修饰对骨髓基质干细胞骨架结构稳定作用及促迁移和对无血清培养诱导细胞凋亡的保护作用研究[J];生物工程学报;2007年04期
，本文编号：2429284
本文链接：https://www.wllwen.com/yixuelunwen/mjlw/2429284.html
最近更新
教材专著
