裂解型腺病毒Ad-E1A的构建以及体外对肝癌细胞的作用

Construction of the oncolytic adenovirus Ad-E1A and its effect on hepatocellular carcinoma cell in vitro

目的构建Ad-E1A裂解型腺病毒载体,研究其对肝癌细胞的裂解作用。方法以QBI-293A细胞基因组DNA为模板,用特异性引物PCR法扩增E1A基因,酶切后连接到pAdTrack-CMV转移质粒上,用PmeI酶对pAdTrack-CMV-E1A线性化后,与pAdEasy-1共转化在BJ5183大肠杆菌中进行同源重组,筛选重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-E1A;再用PacI酶切对重组腺病毒质粒线性化,脂质体转染QBI-293A细胞,收获Ad-E1A腺病毒子。在QBI-293A细胞中多轮感染后获得高效价的病毒。将Ad-E1A腺病毒感染SMMC-7721,并用MTT和流式细胞仪检测Ad-E1A对人肝癌细胞生长和细胞周期的影响。结果获得高滴度的重组裂解型腺病毒Ad-E1A。Ad-E1A感染SMMC-7721后,出现明显的细胞病变。Ad-E1A对人肝癌细胞SMMC-7721的生长有明显抑制,48 h后效果明显。Ad-E1A可直接诱导SMMC-7721细胞的凋亡。结论裂解型腺病毒Ad-E1A构建成功并可以显著抑制人肝癌细胞SMMC-7721的生长,并在肝癌细胞中复制造成细胞裂解,诱导肝癌细胞的凋亡。

Objective To construct the oncolytic adenovirus Ad-E1A and study its the cytolytic effect on human hepatocellular carcinoma （HCC） cell line. Methods The pAdTrack-CMV-E1A was constructed by PCR with QBI-293A genome DNA as template, enzyme digestion and ligation. The pAdTrack-CMV-E1A lineared by PmeI was co-transformed into BJ5183 with pAdEasy-1. The pAdEasy-1-pTrack-CMV-E1A recombined adenovirus vector was lineared with PacI and then transfected into QBI-293A cells. The E1A recombined adenovirus was obtained. The effect on cell growth and cell cycle of human HCC cell line（SMMC-7721） infected with Ad-E1A was determined based on cycotoxicity to SMMC-7721 by MTT assay and RT-PCR analysis. Results The pAdEasy-1- pTrack-CMV-E1A vector was constructed and the high titre E1A recombined adenovirus was obtained. Ad-EIA inhibited SMMC-7721 obviously,especially 24 hours after infection of tumor cells, and induced the SMMC-7721 apoptosis. Conclusion The oncolytic adenovirus Ad-EIA is constructed succefully, which inhibits SMMC-7721 obviously and induces the human hepatoma carcinoma cell apoptosis.