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目的:构建缺失E1A区24 bp,由端粒酶反转录酶基因启动子和缺氧基因启动子调控的增殖腺病毒SG600-IL24,研究人白细胞介素24(interleukin 24,IL24)在肝癌细胞内的表达水平和SG600-IL24的增殖情况及其对肝癌细胞的杀伤作用。方法:利用DNA克隆和重组技术,构建SG600-IL24。ELISA检测IL24在肝癌细胞SMMC-7721、BEL-7404和正常成纤维细胞BJ内的表达。半数组织培养感染剂量(50%tissue culture infectious dose,TCID50)法检测SG600-IL24在3种细胞内48和96 h的增殖情况。MTT法和细胞病理效应(cytopathic effect,CPE)染色法检测SG600-IL24在不同MOI值对3种细胞的杀伤作用。结果:IL24在SMMC-7721和BEL-7404细胞内高表达而在BJ细胞内低表达。感染48和96 h后,SG600-IL24在SMMC-7721、BEL-7404和BJ细胞内分别增殖794和7940倍、622和7 810倍、20和200倍。MTT结果显示,杀伤50%和90%的SMMC-7721、BEL-7404和BJ细胞所需SG600-IL24的MOI值分别为0.3和5,3和20,50和150。CPE染色显示,SG600-IL24对肝癌细胞SMMC-7721和BEL-7404有明显杀伤作用,而对BJ细胞无明显影响,并且该病毒的杀伤能力比增殖腺病毒ZD55-IL24和非增殖腺病毒Ad-IL24强。结论:SG600-IL24高效感染肝癌细胞SMMC-7721和BEL-7404后,病毒增殖活跃,IL24的表达量显著升高。SG600-IL24对此2种肝癌细胞有良好的特异性杀伤作用,而对正常细胞没有明显影响,为应用该病毒治疗肝癌的体内研究建立了基础。
OBJECTIVE: To construct a recombinant adenovirus SG600-IL24 whose deletion was 24 bp in the E1A region and was regulated by the promoter of the telomerase reverse transcriptase gene and the promoter of hypoxia gene. To study the effect of human interleukin 24 (IL24) And the proliferation of SG600-IL24 and its killing effect on hepatoma cells. Methods: SG600-IL24 was constructed by DNA cloning and recombinant technology. ELISA was used to detect the expression of IL24 in hepatoma cells SMMC-7721, BEL-7404 and normal fibroblast BJ. The proliferation of SG600-IL24 at 48 and 96 h in 50% tissue culture infectious dose (TCID50) was detected. MTT assay and cytopathic effect (CPE) staining were used to detect the killing effect of SG600-IL24 on three kinds of cells at different MOI values. Results: IL24 was highly expressed in SMMC-7721 and BEL-7404 cells but was low in BJ cells. After 48 and 96 h infection, SG600-IL24 proliferated 794 and 7940 fold, 622 and 7 810 fold, 20 and 200 fold respectively in SMMC-7721, BEL-7404 and BJ cells. The MTT results showed that the MOI values for SG600-IL24 required for killing 50% and 90% of SMMC-7721, BEL-7404 and BJ cells were 0.3 and 5, 3 and 20, 50 and 150, respectively. CPE staining showed that SG600-IL24 could obviously kill hepatoma cells SMMC-7721 and BEL-7404, but had no obvious effect on BJ cells, and the cytotoxicity of SG600-IL24 was higher than that of adenovirus ZD55-IL24 and non-proliferating adenovirus Ad- IL24 strong. CONCLUSIONS: After highly effective inoculation of SG600-IL24 on SMMC-7721 and BEL-7404 hepatocellular carcinoma cells, the virus proliferates actively and the expression of IL24 is significantly increased. SG600-IL24 has good specific killing effect on these two kinds of hepatocarcinoma cells, but has no obvious effect on normal cells, and establishes the foundation for the in vivo study of applying this virus to treat liver cancer.