腺病毒介导的bak基因诱发肿瘤细胞的凋亡

构建了含ｂａｋ基因的转移载体ｐＣＡ１３－ｂａｋ，并用该载体和含有Ａｄ－５（人腺病毒５型）大部分基因组的重组质凿ｐＢＨＧ１１共转染２９３细胞（转化人胚肾细胞系），分离并提纯缺陷型病毒Ａｄ－ｂａｋ。用该病毒感染ＨｅＬａ细胞（人宫颈癌细胞系），相差显微镜、扫描电子显微镜直接观察到了典型的细胞凋亡形态，联蛋埃Ａｎｎｅｘｉｎ）Ｖ－ＦＩＯＴ７凋亡试剂盒检测到原本位于细胞３膜内侧的磷脂酰丝氨酸向外播转，进一步证

Induction of Apoptosis in Tumor Cells by a Recombinant Adenovirus with bak Gene

Apoptosis, a genetically encoded process of cellular suicide, comprises an intrinsic cellular defence against tumorigenesis. bak (for bcl 2 homologous antagonist/killer) is a new apoptosis inducing gene found recently, which is identified in all tissues examined. Initial work of Farrow, Chittenden and Klefer indicated that bak plays an important role in apoptosis. Successive work of others suggests inactivity of Bak always accompanies the development of tumor cells. In this work, a shuttle vector harboring bak gene, pCA13 bak , was constructed and used to cotransfect into the cell line 293 together with a recombinant plasmid, pBHG11, containing most of adenovirus genome. A mutant adenovirus(delete E1, E3 region)carring bak , Ad bak , was obtained, and purified. Dot blot identified the construction of recombinant virus was successful. HeLa cells were infected by this virus. Some manners were used to identify the changes of HeLa cells including Annexin V FITC staining which made the reversed PS observed directly, microscope observation and others. The characteristic morphology of apoptotic cells was due to Ad bak . The experiment suggests that bak is a strong apoptosis inducing gene, and a potential gene which could be used in gene therapy on tumor.