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目的建立Smad3 WT、Smad3 EPSM及Smad3 3S-A 3种质粒稳转HepG2细胞株,探究单纯转染3种质粒及选择性上调p Smad3C、p Smad3L对HepG2细胞功能的影响。方法采用脂质体转染试剂盒将Smad3 WT(野生型Smad3基因)、Smad3 EPSM(Smad3L区磷酸化位点突变)、Smad3 3S-A(Smad3C区磷酸化位点突变)3种质粒转染至HepG2细胞中,经G418筛选阳性细胞,Western blot法鉴定3种质粒稳转细胞株的转染效率。MTT法检测细胞增殖情况。流式细胞术检测细胞周期及凋亡。结果 Western blot结果显示,转染相应质粒的HepG2细胞高表达目的蛋白,提示稳转细胞株构建成功。MTT结果显示,在缺乏TGF-β_1刺激情况下,转染3种质粒后对HepG2细胞增殖几乎没有影响,TGF-β_1能够诱导稳转细胞株的细胞增殖,且转染Smad3 EPSM质粒的HepG2细胞,较未转染、转染Smad3 WT或Smad3 3S-A质粒的HepG2细胞对TGF-β_1诱导的细胞增殖反应减弱。细胞周期分析显示,TGF-β_1刺激下,转染Smad3 EPSM质粒组G_0/G_1期细胞数明显增多,而转染Smad3 3S-A质粒组G_2/M期细胞数增加明显。细胞凋亡检测显示,TGF-β_1刺激下,较未转染和转染Smad3 WT质粒组,转染Smad3 EPSM质粒组细胞凋亡率明显增加,而转染Smad3 3S-A质粒组细胞凋亡率明显降低。结论成功建立Smad3 WT、Smad3 EPSM及Smad3 3S-A 3种质粒稳转HepG2细胞株,为进一步探讨开发能够经由调控p Smad3C、p Smad3L的药物提供一定的基础。
Objective To establish three HepG2 cell lines stably transfected with three plasmids of Smad3 WT, Smad3 EPSM and Smad3 3S-A, and investigate the effects of transfection of three plasmids alone and selective up-regulation of p Smad3C and p Smad3L on the function of HepG2 cells. Methods Three plasmids of Smad3 WT, Smad3 EPSM, Smad3L and Smad3 3S-A (Smad3C phosphorylation site mutation) were transfected by using Lipofectamine transfection kit In HepG2 cells, positive cells were screened by G418, and the transfection efficiency of the three plasmid-stable cell lines was identified by Western blot. MTT assay cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. Results Western blot results showed that HepG2 cells transfected with the corresponding plasmids highly expressed the target protein, suggesting that the stable transfected cell lines were successfully constructed. MTT results showed that in the absence of TGF-β1 stimulation, three kinds of plasmids transfected HepG2 cells had little effect on proliferation, TGF-β 1 can induce cell proliferation of stable cell lines transfected with Smad3 EPSM plasmid HepG2 cells, Compared with untransfected HepG2 cells transfected with Smad3 WT or Smad3 3S-A plasmid, TGF-β1-induced cell proliferation decreased. Cell cycle analysis showed that TGF-β1-stimulated cells transfected with Smad3 EPSM plasmid group G_0 / G_1 significantly increased the number of cells transfected Smad3 3S-A plasmid group G_2 / M phase cells increased significantly. Apoptosis assay showed that the apoptosis rate of Smad3 EPSM plasmid transfected Smad3 3S-A plasmid group was significantly higher than that of untransfected and transfected Smad3 WT plasmid group under the stimulation of TGF-β1 Obvious reduction. Conclusion The successful establishment of HepG2 cell lines with Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids can provide a basis for the further development of drugs that can regulate p Smad3C and p Smad3L.