细菌内同源重组法制备腺病毒介导TK基因对肝癌细胞的杀伤作用

目的 探讨用细菌内同源重组法制备含TK自杀基因腺病毒对肝癌细胞的杀伤作用.方法 将TK基因自载体中切出,亚克隆至腺病毒穿梭质粒中,形成质粒pAdTrack-GFP-TK,将其PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化BJ5183菌,抽提重组体腺病毒基因组质粒DNA,PacⅠ酶切后转染293细胞包装成腺病毒颗粒,观察其对肝癌细胞的感染效率和目的 基因表达情况及肝癌细胞SMMC-7721感染含TK自杀基因腺病毒后加入丙氧鸟苷(GCV)的生存率和旁观者效应的体外观察.结果 纯化所得腺病毒滴度约为2×1012 efu/mL,当病毒感染复数(MOI)=100时,腺病毒感染SMMC-7721细胞的效率＞90%,实验证实在感染重组体腺病毒的细胞中有相应基因产物的表达,SMMC-7721/Ad-TK细胞对GCV高度敏感,半杀伤浓度(IC50)仅为0.2 μmol/L,HSV-TK/GCV系统在SMMC-7721细胞中存在明显的旁杀伤效应.结论 细菌内同源重组法制备重组腺病毒介导的HSV-TK基因能够成功地在体外整合到肝癌细胞SMMC-7721中,并能高效表达,使其对GCV高度敏感.旁观者效应极大地提高了TK基因抗肿瘤活性,使TK基因制剂实际应用于临床治疗恶性肿瘤成为可能.

Anti-tumor Effect of Adenovirus Vector Generated from Homologous Recombination of Bacteria Mediating TK Gene on Hepatic Neoplasm Cell

OBJECTIVE: To test the anti-tumor effect of adenovirus vector generated from the homologous recombination of bacteria mediating TK gene on hepatic neoplasm cell. METHODS: TK gene was liberated from plasmid and subcloned into shuttle plasmid, which formed the plasmid of pAdTrack-GFP-TK. The pAdTrack-GFP-TK plasmid was then linealized with Pme I and cotransformed into BJ5183 bacterial cells with adenovirus genomic DNA plasmid of pAdEasy-1. The recombinant adenovirus plasmid DNA was digested with Pac I and transfected to the 293 cells to package the recombinant adenovirus particles. The infection rate of the recombinant adenovirus, the target gene expression in the carcinoma cells, the survival rate of the GCV treated hepatic neoplasm cell SMMC-7721 infected with recombinant adenovirus, and the bystander effect were measured. RESULTS: The titre of purified Ad was 2 x 10(12) efu/mL. More than 90% of the SMMC-7721 cells were infected when the MOI reached 100. Target gene expression was detected in the infected cells. The SMMC-7721 cells infected with AdEasy-GFP-TK were very sensitive to the prodrug GCV, with IC50 less than 0.2 pmol/L. Significant bystander effect was observed. CONCLUSION: The constructed AdEasy-GFP-TK virus can efficiently infect the hepatic neoplasm cells and enable the SMMC-7721 cells to express the HSV-TK genes. The bystander effect has improved the anti-tumor effect of the TK gene, which makes the clinical application possible.