hPNAS-4腺病毒载体的构建及其体外抗肿瘤作用

[摘要] 目的：构建携带人的凋亡相关新基因PNAS-4(hPNAS-4)的重组腺病毒，并观察其感染人肺癌A549细胞所引起的hPNAS-4过表达对体外肿瘤细胞凋亡的影响。方法：用RT-PCR从293A细胞中克隆hPNAS-4编码区cDNA，将其酶切后连接至pENTR11载体上，再通过pENTR11与腺病毒骨架载体pAd/CMV/V5-DEST之间的同源重组作用将hPNAS-4基因片段重组至pAd/CMV/V5-DEST上，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；用RT-PCR检测感染细胞中hPNAS-4的过表达情况；通过MTT、流式细胞术及DNA Ladder分别检测感染细胞的增殖与凋亡情况。结果：从293A细胞中中克隆到hPNAS-4全长cDNA并成功构建腺病毒表达载体Ad-hPNAS-4，经测定其滴度为：2.4×108 pfu/ml，感染人肺癌A549细胞后：经RT-PCR测得其mRNA表达明显上调，MTT检测结果为细胞增殖受到明显抑制，流式细胞术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（DNA ladder）；结论：hPNAS-4腺病毒载体感染人肺癌A549细胞后，发现hPNAS-4过表达对肿瘤细胞的生长有明显的抑制和诱导凋亡作用，为今后研究其分子机制以及hPNAS-4腺病毒载体应用于肿瘤动物实验和肿瘤基因治疗提供实验资料。

Construction of Recombinant Adenovirus Vector Carrying hPNAS-4 and its Anti-Tumor Activity in vitro

[Abstract] AIM: To construct recombinant adenovirus vector carrying a novel apoptosis- related gene from human (hPNAS-4). To explore the apoptosis of the tumor cells induced by overpressed hPNAS-4 in human lung carcinoma (A549) cell line via infection of Ad-hPNAS-4. METHODS: RT-PCR was applied to amplify the encoding region of hPNAS-4 gene from the 293A cell line. The ampified gene was cloned into the pENTR11 vector. With the pENTR11-hPNAS-4 plasmid and the backbone plasmid pAd/CMV/V5-DEST, the homologous recombination reaction took place in vitro. The recombination adenovirus plasmid was then generated. The recombinant adenoviruses were packaged and amplified in 293A cells. Ad-hPNAS-4 was used to infect A549 cells. Overexpression of hPNAS-4 in the infected cells was detected by and RT-PCR assay. The proliferation of the infected cells was detected and MTT assay. And the apoptosis was analysed by FCM and DNA ladder. RESULTS: The hPNAS-4 gene was cloned and the recombinant adenovirus vector carrying hPNAS-4 gene was successfully constructed, with a viral titer of 2.4×108 pfu/ml. After A549 cells were infected by Ad-hPNAS-4: RT-PCR 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. CONCLUSION: Overexpression of hPNAS-4 in A549 cells infected by Ad-hPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of A549 cells. And the Ad-hPNAS-4 should be a useful tool for both basic researches on the mechanism study and its application of hPNAS-4 gene to tumor gene therapy.