P19细胞过表达外源Necdin对其细胞增殖的影响

Effect of Overexpressed Exogenous Necdin on the Cell Proliferation of P19 Embryonal Carcinoma Cells

构建PcDNA3.1/necdin真核表达载体并制备稳定过表达Necdin的P19细胞克隆,检测稳定过表达Necdin对P19细胞增殖的影响。从正常培养P19细胞提取RNA反转录成cDNA,以此作为PCR模板扩增得到necdin目的基因,插入PcDNA3.1载体的EcoRⅠ和XhoⅠ位点;脂质体法将构建的真核表达载体PcDNA3.1/necdin转染P19细胞,G418筛选后挑取细胞单克隆,Western印迹鉴定细胞单克隆中Necdin的表达水平。CCK-8法检测过表达necdin对P19细胞增殖的影响。成功构建PcDNA3.1/necdin真核表达载体并获得稳定高表达Necdin的P19细胞克隆,检测发现P19细胞过表达Necdin后其细胞增殖未发生明显变化。P19细胞稳定过表达外源Necdin对其细胞增殖无明显影响。

It was to construct eukaryotic express vector PcDNA3.1/necdin and obtain the monoclonal P19 cell highly expressing necdin, and then detect the effect of overexpressed necdin on the cell proliferation of P19 cell. Total RNA was extracted from normal P19 cells. RT-PCR was used to amplify the aimed segments necdin which was then digested with EcoR Ⅰ and Xho Ⅰ and inserted into a eukaryotic expression plasmid PcDNA3.1 to construct PcDNA3.1/necdin. The constructed vector was transfected into P19 cells through lipofectamine2000-mediated transfer method. The transfected cells were treated with G418 until monoclonal cells appeared. Expression level of Necdin in monoclonal P19 cells was assayed by Western blot. CCK-8 method was utilized to detect the effect of overexpressed necdin on the cell proliferation of P19 cell.Results showed that eukaryotic express vector PcDNA3.1/necdin was successfully constructed and obtained monoclonal P19 cells stably and highly-expressing necdin. Detection of cell proliferation found no obvious change in P19 cells highly expressing necdin. The study showed that overexpressed exogenous necdin have no obvious effect on the cell proliferation of P19 cells.