TGF-β1 shRNA真核表达质粒抑制人肺成纤维细胞TGF-β1的表达

The effect of TGF-β1 expressing inhibition in human lung fibroblast by shRNA of TGF-β1 with eukaryotic expression vectors

目的:观察构建成功的三个针对人转化生长因子β1(TGF-β1)的小发夹RNA(shRNA)真核表达质粒抑制人肺成纤维细胞TGF-β1的表达。方法:将真核表达载体psiSTRIKE-RNAi1、psiSTRIKE-RNAi2、psiSTRIKE-RNAi3和对应的阴性对照载体经酶切鉴定和测序分析后,以Lipofectamine2000介导转染人肺成纤维细胞,48小时后应用实时荧光定量PCR检测人肺成纤维细胞TGF-β1mRNA的表达并用ELISA检测细胞上清液中TGF-β1蛋白质的浓度。结果:重组质粒经酶切和DNA测序证实插入片段的碱基序列与预期设计一致。转染组细胞TGF-β1表达受到明显抑制,以psiSTRIKE-RNAi2的抑制效应最为显著(P<0.01)。结论:三个针对TGF-β1shRNA的重组质粒均能抑制TGF-β1在人肺成纤维细胞中的表达。

Objective：To investigate the effect of transforming growth factor beta 1 （TGF-β1）expressing inhibition induceed by three short hairpin RNA （shRNA） eukaryotic expression plasmids targeting human TGF-β1 gene in human lung fibroblast（HLF）. Methods：Three vectors （ psiSTRIKE- RNAi1, psiSTRIKE- RNAi2, psiSTRIKE- RNAi3） and corresponding negative control plasmids were analyzed by digestion of restriction endonuclease and DNA sequencing, then transferred into HLF by Lipofectamine 2000. After 48 hours, the TGF-β1 mRNA expression in HLF was detected by real time RT-PCR and the TGF-β1 level in the culture supematant was quantitatively determined by ELISA.Results： The DNA sequence inserted in recombinant plasmids was consistent with contemplation. Compared with the control group, the expression of TGF-β1 in the transferred groups was inhibited respectively, and psiSTRIKE-RNAi2 had most significant interference（ P 〈 0.01 ）. Condusion： The three shRNA eukaryotic expressing vectors targeted against human TGF-β1 can inhibit the expression of TGF-β1 in human lung fibroblast.