摘要
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。
Aim To construct three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A stable transfection in HepG2 cell lines to investigate phosphodomains of Snad3( p Smad3 C or p Smad3L),their protein expression and roles in HepG2 cell proliferation,apoptosis and cell cycle. Methods Three plasmids including Smad3 WT( Carry the wild Smad3 gene),Smad3 EPSM( Carry the mutated phosphorylation site in linker region of Smad3 gene) and Smad3 3S-A( Carry the mutated phosphorylation site in C-terminal of Smad3 gene) were respectively transfected into HepG2 cells by using a liposome transfection reagent. Verification of positive cells was done by screening with G418 via co-culture. Transfection efficiency was determined by Western blot. Cell proliferation was induced by exogenous TGF-β_1in the respective stably transfected HepG2 cell lines. Cell proliferation was monitored by MTT. Cell cycle and apoptosis were determined by flow cytometry( FCM). Results There was elevated protein expression of the respective phospho-domain sites in the stably transfected HepG2 cells for Smad3WT( C-terminus and Linker),Smad3 EPSM( C-terminus) and Smad3 3S-A( Linker),which indicated successful stable transfection of HepG2 cell lines. The results from MTT experiment showed that TGF-β_1could induce proliferation of HepG2 cells with or without the transfection of Smad3 WT,Smad3 EPSM and Smad33S-A plasmids,meanwhile transfected Smad3 EPSM plasmids could significantly inhibit proliferation of HepG2 cells induced by TGF-β_1, and transfected Smad3 3S-A plasmids accelerate proliferation of HepG2 cells induced by TGF-β_1. Cell cycle analysis showed that the G_0/G_1 phase of HepG2 cells with stable transfection of Smad3 EPSM plasmid increased compared with HepG2 cells with or without stable transfection of Smad3 WT plasmid,meanwhile the G_2/ M phase of HepG2 cells with stable transfection of Smad3 3S-A plasmid increased. Compared with Smad3 WT transfected cells, apoptosis in Smad3 EPSM transfected cells was markedly increased,while that of Smad3 3SA transfected cells decreased. Conclusions The three plasmids of Smad3 WT,Smad3 EPSM and Smad3 3SA stably transfected HepG2 cell lines have been successively constructed. The construction of three plasmids transfected HepG2 cell lines provides the research foundation for studying medical as well as possible regulatory mechanism of p Smad3 C / p Smad3 L.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2016年第6期825-831,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81374012
81573652)
