腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

The Growth-inhibitory Effect of Recombinant OSM-Adenovirus on A375 Melanoma Cells

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。

Objective： To construct recombinant adenovirus harboring OSM gene and to study its growth-inhibitory effects on A375 melanoma cells.Methods： In order to obtain the recombinant OSM Adenovirus vector,PCR technique was used and PEGZ-OSM was used as the template.Then the recombinant Ad-OSM was obtained by gene recombination and in vitro packaging technique.A375 cells were infected with Ad-OSM and RT-PCR was used to detect the transcription of OSM in the cells.Western blot was used to detect the expression of OSM in the cells.Cell growth inhibition was detected by MTT assay.Morphologic changes were observed by light microscope and fluorescence microscope.The cell cycle and apoptosis were detected by flow cytometry and RT-PCR was used to detect the transcription of Bax and Bcl-2.Results： Ad-OSM was proved in correct construction by gene sequence and RT-PCR results.Expression of OSM was identified by RT-PCR.By microscope and flow cytometry displayed the apoptosis of the melanoma cells positively expressing OSM.Additionally,the mRNA of Bax was up-regulated and mRNA of Bcl-2 was down-regulated in the cells by RT-PCR.Conclusion： Ad-OSM was proved in correct construction.Ad-OSM can inhibit the growth of A375 cells and induce cell apoptosis,which may result in changing Bax and Bcl-2 expression.