新型增殖性腺病毒的构建及对肝癌细胞的抑制

Construction of a new type replication-selective adenovirus CNHK600-p53 and its inhibitory effect on hepatocellular carcinoma cells in vitro

目的 构建携带p53基因新型增殖性腺病毒CNHK600-p53,研究其对肝癌细胞株抑制效应是否优于Ad-p53.方法 PCR扩增p53基因,利用酶切连接方法 将其插入CNHK600载体,PCR鉴定.经293细胞包装成病毒,抽提病毒DNA,PCR鉴定.氯化铯密度梯度离心法纯化病毒,TCID50 方法 测病毒滴度.病毒增殖实验检测病毒在不同细胞增殖能力.四甲基偶氮唑盐（methyl-thiazolyl tetrazolium assay,MTT）法观察CNHK600-p53、Ad-p53两种病毒分别对肝癌细胞株的抑制率.结果 成功构建新型增殖性腺病毒载体CNHK600-p53;293细胞包装成病毒,PCR方法 鉴定无野生型病毒存在;病毒滴度为1.99×10^10pfu/ml;CNHK600-p53在肝癌细胞HepG2、SMMC-7721内复制能力明显高于正常肝细胞HEL-1和L02.对6种肝癌细胞（PLC/PRF5、SMMC7721、HepaG2、BEL-7402、BEL-7404、QGY-7703）而言,随着MOI值的不断增高.其对肝癌细胞的抑制作用也不断增强;在相同MOI情况下,CNHK600-p53组较Ad-p53组的细胞抑制率高（P＜0.05）.当细胞抑制率达到80%以上时.两组之间差异无统计学意义（P＞0.05）.结论 利用新型增殖性腺病毒CNHK600一p53较 Ad-p53能更有效的抑制肝癌细胞,可能成为肝癌的基因治疗更有效的一种基因治疗手段.

Objective To construct a new replicating adenovirus vector CNHK600-p53 which carried the anti-tumor gene p53 and determine whether its inhibitory effect on hepatoceltular carcinoma cell lines is better than Ad-p53 or not.Methods The p53 gene was amplified by PCR and inserted into plasmid CNHK600 genome to obtain the new replicating adenovirus vector CNHK600-p53 by enzymatic digestion and ligation methods.After packaging in 293 cells,the new replicating adenoviruses CNHK600-p53 DNA was extracted and identified by PCR.Viruses were purified by CsCl gradient purification and Viruses titer was estimated from TCID50.MTT method was used to observe the inhibition rates of hepatocellular carcinoma cells by CNHK600-p53 and Ad-p53 to.Results The new replicating adenovirus vector CNHK600-p53 was sucessfully constructed.There was no wild type virus after packaging in 293 cells and viruses titer was 1.99 × 10^（10） pfu/ml.The replication ability of CNHK600-p53 was better in hepatocellular carcinoma cell lines HepG2 and SMMC-7721 cells than in normal cell lines HEL-1and L02.To six hepatocellular carcinoma cell lines（PLC/PRF5,SMMC7721,HepaG2,BEL-7402,BEL-7404 and QGY-7703）,MOI was higher and the inhibitory effect was stronger.To every cell line,the inhibitory rate of CNHK600-p53 was higher than that of Ad-p53 at the same MOI（P〈0.05）.However,when inhibitory rate was larger than 80%,there are no significant difference between two groups（P〉0.05）.Conclusion CNHK600-p53 is more effective to inhibit hepatocellular carcinoma cells than Ad-p53 and might serve as a new tool for hepatocellular carcinoma gene therapy.