Smad泛素化调节因子2siRNA的筛选及转染原代肾小管上皮细胞条件的优化  

Screening and Transfection Condition Optimization of Smurf2siRNA in Renal Tubular Epithelial Cells

作　　者：赵海焦[1] 高磊[1] 陆彦[1] 田娅慧[1] 贾志华[1] 田海燕[2] 吴国娟[1] 张中文[1] 

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京观赏动物医院,北京100029

出　　处：《中国畜牧兽医》2014年第1期19-24,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31172362;31201949;31372485);北京市人才强教深化计划-教学名师项目(PHR201106219)

摘　　要：试验旨在优化Smad泛素化调节因子2（Smurf2） siRNA最佳转染条件，筛选最佳siRNA干扰片段，进而实现对Smurf2基因的瞬时沉默，为研究Smurf2基因在马兜铃酸肾病（aristolochic acid nephrohathy，AAN）中的作用奠定基础。本研究通过培养小鼠原代肾小管上皮细胞（renal tubular epithelial cells，RTECs），并以脂质体LipofectamineTM 2000为转染介质，将Smurf2 siRNA转染入RTECs；通过观察绿色荧光的表达量及Real-time PCR反应优化转染条件，转染后Real-time PCR检测mRNA表达抑制率。CCK-8法检测Smurf2 siRNA复合物对RTECs活性的影响；同时，用Real-time PCR和Western blotting检测不同位点Smurf2 siRNA对Smurf2 表达的影响。结果显示,当Lipofectamine TM 2000与siRNA比例为1.5 μL∶30 pmol时，转染效率最高，为70%～80%；Smurf2-619 siRNA干扰效果最明显；与正常组相比，转染siRNA组的Smurf2蛋白表达水平显著下降（P〈0.05）。最终确定了Smurf2 siRNA最佳转染条件，其中Smurf2-619 siRNA对Smurf2 表达的抑制率最高。To lay the foundation for investigating the role of smurf2 in aristolochic acid nephropathy,we optimized the best transfection condition of Smurf2 siRNA,and screenned the best siRNA fragment,then realized the Smurf2 transient gene silencing. We cultured renal tubular epithelial cells （RTECs）,LipofectamineTM 2000 was taken for transfection medium,transfecting Smurf 2 siRNA into RTECs,the transfection condition was optimized by observing expressions of green fluorescences and undertaking Real-time PCR reactions.After transferring the Smurf2 siRNA, mRNA inhibition ratios were detected by Real-time PCR.We detected the effects of compound Smurf2 siRNA on RTECs activity by CCK-8;at the same time,we detected the expression of Smurf2 under different sites of Smurf2 siRNA by Real-time PCR and Western blotting. The results showed that the best transfection condition was 1.5 μL LipofectamineTM 2000 and 30 pmol siRNA,the transfection efficiency was 70% to 80%.Smurf2-619 siRNA was proved with the most obvious interference effect.Smurf2 protein levels decreased significantly in the group of Smurf2 siRNA compared with the normal group （P〈0.05）.We determined the best transfection condition of Smurf2 siRNA, Smurf2-619 siRNA had the highest inhibition rate for the expression of Smurf2.

关 键 词：Smad泛素化调节因子2 转染 SIRNA 肾小管上皮细胞 优化 

分 类 号：Q78[生物学—分子生物学]