摘要目的 构建CD以及VP22-CD基因工程化载体,并探讨其在体外对胶质瘤细胞的杀伤作用.方法 利用聚合酶链式反应(PCR)得到CD以及VP22基因片段;利用In-fusion基因克隆方法将得到的基因片段插入到pEGFP-N1载体中,构建CD以及VP22-CD基因工程化载体;利用脂质体转染的方法将构建的基因工程化载体转染到胶质瘤细胞C6及U87细胞中,并通过体外光镜及荧光显微镜观察转染率;利用MTT比色法评价加入前药5-氟胞嘧啶后对转染后胶质瘤细胞的杀伤率.结果 (1)将CD基因或VP22-CD融合基因插入到pEGFP-CD载体中,构建得到pEGFP-CD以及pEGFP-VP22-CD载体.(2)pEGFP-CD及pEGFP-VP22-CD载体可转染入C6或U87胶质瘤细胞,转染率分别为2.5％和3.7％以及2.0％和4.1％.(3)在C6或U87细胞中转染pEGFP-CD或pEGFP-VP22-CD载体并加入前药5-氟胞嘧啶后,细胞的存活率分别为(30.36±0.63)％和(11.75±1.01)％以及(28.78±3.62)％和(18.26±2.27)％,与转染pEGFP-N1组相比差异均有统计学意义(均P＜0.05).(4)在C6或U87细胞中加入前药5-氟胞嘧啶后,转染pEGFP-VP22-CD组与转染pEGFP-CD组相比差异均有统计学意义(均P＜0.05).结论 CD/5-FC系统在体外对恶性胶质瘤细胞具有显著的杀伤作用和旁观者效应;并且穿梭蛋白VP22可进一步促进系统对细胞的杀伤作用.

abstractsObjectives To construct CD and VP22-CD genetically engineered vectors and to investigate their killing effects on glioma cells in vitro.Methods CD and VP22 gene segments were obtain using polymerase chain reaction (PCR).The obtained gene fragments were inserted into pEGFP-N1 vectors using in-fusion gene cloning method.CD and VP22-CD gene engineering vectors were constructed.The constructed gene engineering vectors were transfected into the glioma cells C6 and U87 cells using the liposome transfection method,and the transfection efficiency was observed through in vitro light microscope and fluorescence microscope.The kill rate of glioma cells after transfection was assessed after adding the prodrug 5-fluorocytosine using MTF colorimetric method.Results (1) CD gene or VP22-CD fusion gene was inserted into the pEGFP-CD vectors,constructing and obtaining pEGFP-CD and pEGFP-VP22-CD vectors.(2) The pEGFP-CD and pEGFP-VP22-CD vectors were transfected into C6 or U87 glioma cells.The transfection rates were 2.5％ and 3.7％,2％ and 4.1％,respectively.(3) Transfection of pEGFP-CD or pEGFP-VP22-CD vectors in the C6 or U87 cells and after adding prodrug 5-fluorocytosine,the survival rates were 30.36±0.63％ and 11.75±1.01％,28.78 ±3.62％ and 18.26±2.27％,respectively.Compared with the transfection of pEGFP-N1 group,there was significant difference (all P ＜ 0.05).(4) After adding prodrug 5-fluorocytosine in C6 or U87 cells,There was significant difference between the transfection of pEGFP-VP22-CD group and the transfection of pEGFP-CD group (all P ＜ 0.05).Conclusions The CD/5-FC system has significant killing effect and bystander effect on the malignant glioma cells in vitro,and shuttle protein VP22 can further promote the killing effect of the system on cells.