RNAi干扰WT1和Pax2表达对逆转肾小管上皮间充质转化的作用  

Reverse Effect on Renal Tubular Epithelial to Mesenchymal Transition by RNAi Silencing WT1 and Pax2 Expression

作　　者：黄彬[1] 姜傥[1] 皮蕾 滕军旗[1] 

机构地区：[1]中山大学附属第一医院检验医学部,广东广州510080 [2]广州市妇女儿童医疗中心检验科,广东广州510120

出　　处：《中山大学学报（医学科学版）》2011年第4期442-448,510,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30370661);广东省自然科学基金(31683)

摘　　要：【目的】探讨抑制肾小管上皮间充质转化(EMT)过程中胚胎发育关键基因WT1和Pax2的表达对EMT的逆转作用。【方法】采用RNAi技术分别抑制WT1和Pax2。分别构建pshRNA-WT1和pshRNA-Pax2表达载体,采用脂质体转染技术,使质粒瞬时转染NEK52E细胞后用10ng/mLIL-1α刺激细胞,分别提取不同时间点细胞的RNA和蛋白质,采用RT-PCR和Western blot检测细胞WT1、Pax2、Snail、上皮细胞标志E-cadherin和间充质标志α-SMA的mRNA和蛋白质的表达,并观察NRK52E细胞的形态。【结果】构建的pshRNA-WT1和pshRNA-Pax2表达载体可在转染细胞内发挥RNAi作用抑制WT1和Pax2基因的表达,抑制率分别为80.4%和82.7%。分别抑制WT1和Pax2基因后EMT过程受阻,α-SMA和Snail的表达显著减少,E-cadherin的表达无显著性变化,细胞形态未发生明显的成纤维化样改变。【结论】WT1和Pax2基因是EMT中的关键基因,分别抑制胚胎发育关键基因WT1和Pax2均可使EMT受阻。阻断WT1和Pax2的表达有望中止EMT及肾纤维化的发生。[Objective] To explore the reverse effect on renal tubular epithelial to mesenchymal transition by RNA interference（RNAi） silencing the embryonic genes WT1 and Pax2 expression.[Methods] RNAi was used to inhibit WT1 and Pax2.WT1 shRNA expression vector pshRNA-WT1-452 and pshRNA-WT1-283,Pax2 shRNA expression vector pshRNA-Pax2-983 and pshRNA-Pax2-302 were constructed.Plasmids were transfected into NEK52E cells with cationic liposome,and then 10 ng/mL IL-1α was added to stimulate NRK52E cells.RNA and protein were extracted at different time points respectively,and WT1,Pax2,Snail,the epithelial marker E-cadherin,the mesenchymal marker α-SMA mRNA and protein were detected using RT-PCR and Western blot analysis;the morphology of cells was observed.[Result] pshRNA-WT1 and pshRNA-Pax2 specifically and efficiently inhibited WT1 and Pax2 expression in NEK52E cells by RNAi,the repression rates were 80.4% and 82.7%,respectively.Repressing WT1 and Pax2 using RNAi blocked EMT in NRK52E cells treated with IL-1α,which was evidenced by suppressed α-SMA,Snail expression,restored E-cadherin expression,and the normal cell morphology.[Conclusion] Our experiments suggest that WT1 and Pax2 expression in tubular epithelial cells play an important role in promotion of EMT,and there may be therapeutic value in silencing WT1 and Pax2 so as to prevent or to reverse renal fibrosis.

关 键 词：RNAI 上皮间充质转化 肾小管上皮细胞 

分 类 号：R446[医药卫生—诊断学]