CXCR4启动子的条件复制型腺病毒对肺癌细胞的靶向杀伤作用

摘要

[ ABSTRACT] AIM:To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically.METHODS:Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP.The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP.PCR was used to detect the target gene fragments, and the viral titer was determined.A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR.CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL.CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay.RE-SULTS:The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully.Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus.PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome.The virus in the supernatant reached a titer of 1 ×1013 PFU/L.The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group.Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days.CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.%目的：构建CXCR4启动子的条件复制型腺病毒载体CRAd-CXCR4-GFP，探究CRAd-CXCR4-GFP对肺癌细胞的靶向杀伤效应。方法：PCR法扩增人CXCR4-E1A基因并克隆至穿梭质粒pDC316-GFP，将骨架质粒pBHG-lox-E1，3Cre和重组质粒pDC316-CXCR4-GFP共转染293细胞，产生重组腺病毒CRAd-CXCR4-GFP并扩增，扩增后进行PCR鉴定和腺病毒滴度测定；real-time PCR检测5种肺癌细胞株CXCR4的mRNA表达，筛选出表达最高的A549细胞；将CRAd-CXCR4-GFP和Ad-NULL分别转染A549细胞，检测两者转染后CXCR4启动子活性和腺病毒复制数；将CRAd-CXCR4-GFP和Ad-NULL分别转染A549细胞和16HBE细胞，流式细胞术检测各组细胞凋亡情况，CCK-8检测各组细胞活性。结果：成功构建重组质粒pDC316-CXCR4-GFP，与骨架质粒pBHG-lox-E1，3Cre共转染293细胞后第11天可见片状绿色荧光；PCR表明目的基因CXCR4-E1A已成功整合在重组腺病毒基因组中；测定重组腺病毒滴度为1×1013 PFU／L；CRAd-CXCR4-GFP转染A549细胞后E1A的mRNA和E4表达较Ad-NULL组明显上升；与Ad-NULL组和空白对照组相比，CRAd-CXCR4-GFP组前4 d A549细胞凋亡率和活性无明显差异，第5天细胞凋亡率明显增加，细胞活性明显降低，各组间5 d内16 HBE细胞的细胞凋亡率和细胞活性均无明显变化。结论：成功构建条件复制型腺病毒表达载体CRAd-CXCR4-GFP，该载体对肺癌细胞具有靶向杀伤作用。