人肾系膜细胞Smad-7稳定表达细胞系的筛选及其拮抗TGF-β1作用

Construction of eukaryotic expression plasmid of Smad-7 and study on its function as TGF-β1 inhibitor

目的构建Smad-7基因真核表达载体并筛选获得其稳定表达细胞系，观察Smad-7拮抗TGF-β1对人肾系膜细胞（HMC）的影响。方法用PCR方法从胎肝cDNA文库扩增Smad-7编码序列，将其构入真核表达载体pcDNA3．1／myc-his-B（-），酶切及测序鉴定重组质粒。重组质粒转染HMC细胞，G418筛选获得Smad-7的稳定表达细胞克隆，运用Western Blotting验证目的基因表达。运用MTT和流式细胞术检测稳定表达Smad-7对TGF-β1诱导的拮抗作用。结果酶切和测序证实目的基因被克隆入表达载体，Western Blotting证实目的基因成功表达，获得稳定表达Smad-7的细胞克隆；Smad-7过表达可明显缓解TGF-β1对HMC细胞的生长抑制、细胞周期G1阻滞作用，并可降低TGF-β1对HMC细胞的凋亡诱导作用。结论成功构建Smad-7真核表达载体并获得其稳定表达细胞克隆，过表达Smad-7可拮抗TGF-β1对HMC细胞的影响。

Objective To construct eukaryotic expression vectors stably expressing Smad-7 and study its function as TGF-β1 inhibitor. Methods Smad-7 genes were obtained by PCR from fetal liver cDNA library and inserted in to eukaryotic expression plasmid pcD- NA3.1/myc-his-B （ - ）. The constructed plasmids containing right sequence of target genes were transfected into human mesangial cells by lipofectamine. The ceils were selected by Gd-18, the expression of Smad-7 was detected by Western-blotting. Cell proliferation was detected by MTF method, and cell cycle was detected by Flow Cytometer to study its function as TGF-β1 inhibitor. Results We successfully con- struct Smad-7 expression plasmid. Smad-7 can inhibit the effects of TGF-β1 induced G1 cell cycle block and apoptosis. Conclusion The eukaryotic expression vector of Smad-7 has been successfully constructed. Over-expression of exogenous Smad-7 can inhibit the effects of TGF-β1 induced G1 cell cycle block and apoptosis.