慢病毒介导的TGFβ-R2 shRNA抑制人LECs细胞TGFβ-R2的表达
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上海市卫生局青年科研项目(No.2009Y126)


Inhibition of lentivirus-TGFβ-R2 shRNA on the expression of TGFβ-R2 in human lens epithelial cells
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Shanghai Municipal Health Bureau Youth Projects(No.2009Y126)

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    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。

    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.

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李贞,徐庆,张慧.慢病毒介导的TGFβ-R2 shRNA抑制人LECs细胞TGFβ-R2的表达.国际眼科杂志, 2014,14(1):9-12.

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  • 收稿日期:2013-10-08
  • 最后修改日期:2013-12-09
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  • 在线发布日期: 2013-12-23
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