在结肠癌细胞内高效表达内皮抑素的增殖型腺病毒载体的构建与鉴定

Construction and identification of replication-competent adenovirus highly expressing human endostatin in colon cancer cells in vitro

目的 :构建携带内皮抑素基因的增殖型腺病毒载体 ,寻找结肠癌的有效治疗方法。方法 :通过 RT- PCR克隆内皮抑素基因 ,2 93细胞内分别重组携带内皮抑素基因的增殖型 (CNHK2 0 0 - h E)和增殖缺陷型 (Ad- h E)腺病毒 ,分别体外感染结肠癌细胞 HT- 2 9后比较两者在细胞病理效应、病毒增殖和内皮抑素表达量方面的差异。 结果 :CNHK2 0 0 - h E感染结肠癌细胞96 h后可增殖 2 0 0 0多倍 ;感染后 7d肿瘤细胞死亡率达 96 .2 % ,而对正常肝细胞基本无杀伤作用 ;随着感染时间的延长 ,肿瘤细胞培养上清液中内皮抑素表达量不断增加 ,第 5天达 1 0 90 ng/ ml,显著高于 Ad- h E感染组 (P<0 .0 1 )。 结论 :携带内皮抑素基因的增殖型腺病毒 CNHK2 0 0 - h

Objective:To construct a replication competent adenovirus vector carrying human endostatin gene,looking for an effective method for the therapy of colon cancer. Methods: Endostatin gene was cloned by RT PCR and then the replication competent (CNHK200 hE) or replication deficient (Ad hE) adenovirus carrying endostatin gene were reconstructed in 293 cells.Their cytopathic effect and endostatin expression were compared when they were used to infect HT 29 cells. Results: CNHK200 hE could be replicated by more than 2 000 times in colon cancer cells 96 h after infection.The death rate of tumor cells reached 96.2% 7 d after infection while there was no harm to the normal liver cells.The endostatin expression in CNHK200 hE group was increased with the time and reached 1 090 ng/ml 5 d after infection,which was markedly higher than that in Ad hE group at the same time. Conclusion: CNHK200 hE, a replication competent adenovirus mediating human endostatin gene, has the ability to replicate in colon cancer cells and efficiently express the endostatin.