新城疫病毒HN基因诱导人肺癌细胞SPC-A1凋亡的作用机制

为了探索新城疫病毒血凝素神经氨酸酶(HN)基因对人肺癌细胞SPC-A1的作用及机制，将含有HN基因的重组质粒pVHN经脂质体介导转染人肺癌细胞SPC-A1，通过MTT方法检测细胞活力；采用吖啶橙/溴化乙锭（AO/EB）染色分析肿瘤细胞凋亡；罗丹明123和DCFA染色测定线粒体跨膜电位（ΔΨm）和活性氧水平变化；流式细胞仪分析MHCⅠ分子表达； 底物染色反应检测caspase-3活性结果重组质粒pVHN转染人肺癌细胞SPCA1 48 h后，细胞活力明显降低； AO/EB染色可见明显的细胞凋亡形态学变化；与空质粒对照相比，线粒体ΔΨm下降(Ｐ<0.01)，活性氧水平升高(Ｐ<0.05)；细胞表面MHC-Ⅰ分子表达上调(P>0.05)；caspase-3活性增强(Ｐ<0.01). 以上结果提示，新城疫病毒HN基因能够上调SPC-A1细胞表面MHC-Ⅰ分子表达，并通过上调ROS水平，下调线粒体ΔΨm，进而激活caspase-3，最终诱导人肺癌细胞凋亡.

Mechanisms of Apoptosis in Human Lung Carcinoma Cells SPC-A1 Induced with Hemagglutinin-neuraminidase(HN) Gene from Newcastle Disease Virus

To investigate the effect of hemagglutinin-neuraminidase(HN) gene from Newcastle disease virus on human lung carcinoma cells SPC-A1, the recombinant plasmid pVHN was introduced into human lung carcinoma cells SPC-A1 byliposome-mediated transfection. The viability of SPC-A1 cells was determined by MTT staining and apoptosis was detected by AO/EB staining. The alteration of mitochondrial transmembrane potential and ROS level of the cells was detected by flow cytometry (FCM) with rhodamine 123 and DCFA staining.The expression of MHC-Ⅰ was detected by FCM and the activation of caspase-3 was assayed by its substrate color reaction. Viability of SPC-A1 cells was decreased significantly and recombinant plasmid could lead to obviously morphological apoptotic changes of SPC-A1 cells by introduction of pVHN into SPC-A1 cells for 48 hours. Mitochondrial transmembrane potential was decreased(Ｐ<0.01) , ROS level of the cells was increased (Ｐ<0.05), theexpression of MHC-Ⅰ(P>0.05) and caspase-3 activity (Ｐ<0.01)was increased, compared with control cells.The above results showed increase of MHC-Ⅰ expression andsignificant apoptosis in SPC-A1 cells can be induced by recombinant plasmid pVHN. Apoptosis may be resulted from the increase of ROS level, the decrease of mitochondrial transmembrane potential and activation of Caspase-3.