<i>Noxa</i>基因的克隆及其诱导A549细胞凋亡的研究

陈登榜; 曹康; 杨靖; 潘渠; 陈恬; 李晋川; 王昕; 殷建华; 邹毅; 魏常友

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

摘要

摘要: 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。

Abstract: Objective To clone the Noxa gene and to observe the apoptosis of A549 cells transfected with the recombinant plasmid of pcDNA-Noxa. Methods The Noxa gene was obtained by PCR, and was cloned into pcDNA3.1(-). A549 cells were transfected with the recombinant plasmid of pcDNA-Noxa. Western blot analysis was performed to determine the overexpression of Noxa. A549 cells were stained with Hoechst 33258 to 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 A549 cells. Conclusion Noxa has exhibited potential pro-apoptotic activity against A549 cells. This study is a foundation for further research into pro-apoptotic activity of Noxa gene.

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