hPNAS-4腺病毒载体的构建及其体外抗肿瘤作用

Construction of recombinant adenovirus vector carrying hPNAS-4 and its anti-tumor activity in vitro

用RT-PCR从A293细胞中克隆hPNAS-4编码区cDNA,将其酶切后连接至pEN-TRll载体上,再通过同源重组作用将hPNAS-4基因片段重组至腺病毒骨架载体上,构建腺病毒重组质粒,最后经293细胞的包装扩增后得到携带hPNAS-4基因的重组腺病毒;将重组腺病毒体外感染人肺癌A549细胞;RT-PCR测得感染细胞中hPNAs-4表达明显上调,MTT测得细胞增殖受到明显抑制,流式细胞仪测得细胞凋亡率明显升高,琼脂糖凝胶电泳显示其基因组DNA有明显的梯状条带.以上结果表明hPNAS-4过表达对A549肿瘤细胞的生长有明显的抑制和诱导凋亡作用.

RT-PCR was applied to amplify the encoding region of hPN^1 AS-4 gene from the A293 cell line. The ampified gene was cloned into the pENTR11 vector and then subcloned into adenoviral backbone vector with the homologous recombination reaction. The recombination adenovirus plasmid was generated. The recombinant adenoviruses Ad-hPNAS-4 were packaged and amplified in A293 cells. AdhPNAS-4 was used to infect A549 cells. RT-PCR assay showed that the expression of hPNAS-4 at the mRNA level was up-regulated significantly; MTT assay showed that the proliferation was inhibited; Flow cytometry showed that higher apoptosis rate was observed; Genome DNA electrophoresis showed the characteristic DNA ladder of apoptosis. All the results suggest that the overexpression of hPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of A549 cells.