Noxa基因的克隆及其诱导A549细胞凋亡的研究

Apoptosis of A549 Cells Induced by Cloned Noxa Gene

目的克隆人Noxa基因,构建真核表达质粒pcDNA-Noxa,将重组质粒pcDNA-Noxa转染人肺癌A549细胞株,并观察重组质粒pcDNA-Noxa诱导A549细胞凋亡的作用。方法使用RT-PCR的方法扩增出Noxa基因,克隆至真核表达质粒pcDNA(-),构建出重组质粒pcDNA-Noxa。将重组质粒pcDNA-Noxa转染人肺癌A549细胞株,使用Western blot的方法检测Noxa基因的过表达;使用Hoechst 33258染色,观察A549细胞的凋亡;使用MTT法检测细胞活力。结果经酶切和测序鉴定,重组质粒pcDNA-Noxa构建成功。Western blot检测出Noxa基因的过表达;Hoechst 33258染色和MTT实验发现:重组质粒pcDNA-Noxa能明显诱导A549细胞出现凋亡现象。结论本实验初步探索了Noxa基因的促凋亡作用,为以后进一步深入研究Noxa基因的促凋亡功能基础。

Objective To clone the Noxagene and to observe the apoptosis of A549cells transfected with the recombinant plasmid of pcDNA-Noxa.Methods The Noxa gene was obtained by PCR,and was cloned into pcDNA3.1(-).A549cells were transfected with the recombinant plasmid of pcDNA-Noxa.Western blot analysis was performed to determine the overexpression of Noxa.A549cells were stained with Hoechst 33258to observe the apoptosis.Results The recombinant plasmid of pcDNA-Noxa was successfully constructed evidenced by endonuclease digestion and sequence analysis.The overexpression of Noxa was identified using Western blot analysis.The recombinant plasmid of pcDNA-Noxa induced apoptosis of A549cells.Conclusion Noxa has exhibited potential pro-apoptotic activity against A549cells.This study is a foundation for further research into proapoptotic activity of Noxagene.