[关键词]
[摘要]
目的：观察转化生长因子β-R2特异性短发夹RNA(shRNA)对人晶状体上皮细胞TGFβ-R2 表达的影响。
方法：根据小干扰RNA(siRNA)的设计原则，针对转化生长因子β-受体2基因序列特征构建特异性shRNA(RNAi)载体，与TGFβ-R2过表达载体共转染293T细胞，经Western blot筛选出有效RNAi载体后，进行扩增、纯化、滴度测定，并转染培养的人LECs，qPCR检测TGFβ-R2 siRNA慢病毒载体对TGFβ-R2 mRNA表达的影响。
结果：经Western blot筛选，TGFβ-R2/GV115RNAi#1对目的基因的表达敲减效果最明显，慢病毒包装，滴度为8E+8 TU/mL，感染人LECs细胞后，对TGFβ-R2基因有显著的敲减效果，达到78.1%(P<0.05)。
结论：TGFβ-R2 RNAi载体构建成功，并且能抑制人晶状体上皮细胞TGFβ-R2 mRNA的表达。
[Key word]
[Abstract]
AIM: To observe the suppressing effect of specific small hairpin RNA(shRNA)on TGFβ-R2 expression in human lens epithelial cells(LECs).
METHODS: Specific shRNA expression vector was constructed according to the design principles of TGFβ-R2 mRNA human GeneBank siRNA synthesis and transfected into cultured 293T with TGFβ-R2 over expression vector. After effective RNAi vector was selected by Western blot, then amplification, purification, and titration. lentivirus-TGF beta-R2 shRNA was transfected into LECs, then TGFβ-R2 mRNA expression in these cells was detected by the real-time fluorescence quantitative PCR detecting system(qPCR).
RESULTS: TGFβ-R2/GV115RNAi#1 target gene expression knockdown effect is the most obvious; the titer of packaging lentivirus is 8E+8 TU/mL; the knockdown effect is significant, the interfering efficiency is 78.1%(P<0.05).
CONCLUSION: TGFβ-R2 RNAi vector was successfully constructed, and can inhibit the expression of TGFβ-R2 mRNA in human LECs.
[中图分类号]
[基金项目]
上海市卫生局青年科研项目(No.2009Y126)
