非增殖型和靶向增殖型腺病毒携带TRAIL基因治疗肝癌的实验研究

GENE THERAPY OF HEPATOCELLULAR CARCINOMA USING REPLICATION-DEFECTIVE AND TARGETED TUMOR-SPECIFIC REPLICATION-COMPETENT ADENOVIRUS CARRYING HUMAN TRAIL GENE IN VITRO

目的比较携带人TRAIL基因的增殖型腺病毒(CNHK500-hTRAIL)和非增殖型腺病毒(Ad-hTRAIL)对TRAIL基因的表达以及对SMMC-7721肝癌细胞的杀伤能力。方法通过病毒增殖实验评估增殖型腺病毒CNHK500-hTRAIL的选择性增殖能力。通过MTT实验,评估增殖型腺病毒CNHK500-hTRAIL以及非增殖型腺病毒Ad-hTRAIL对人正常肝细胞株L02、人肝癌细胞株SMMC-7721的杀伤能力。采用ELISA法检测CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721肝癌细胞后TRAIL基因的表达情况。以及通过流式细胞术(FCM)检测其对细胞早期凋亡的影响。结果CNHK500-hTRAIL能选择性地在SMMC-7721细胞内大量增殖,感染96 h后增殖达225137倍,在极低的MO I值(MO I=0.1)即可大量杀伤SMMC-7721细胞,明显强于Ad-hTRAIL,而对L02细胞无明显杀伤。CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721细胞后,其TRAIL基因表达量,前者是后者的近10倍;CNHK500-hTRAIL可选择性地诱导SMMC-7721细胞早期凋亡,其能力显著高于Ad-hTRAIL。结论靶向增殖型腺病毒载体携带TRAIL基因对肿瘤细胞的杀伤能力和目的基因的表达,明显优于传统的非增殖型腺病毒载体,应用前景广阔。

Objective To evaluate the therapeutic effect and the expression level of a tumor-specific replication-competent adenovirus and a replication-defective adenovirus expression human soluble TRAIL（hTRAIL） gene on hepatocellular carcinoma cell line SMMC-7721 in vitro. Methods Virus replication assay was performed to evaluate the selective replication ability of CNHK500- hTRAIL. The cytotoxicity of replication- competent adenovirus CNHK500-hTRAIL and replication-defective adenovirus Ad-hTRAIL were evaluated by MTT in HCC cell line SMMC-7721 and human normal hepatocyte line L02. ELISA assay were used to determine the expression level of TRAIL. Flow Cytometry （ FCM ） was used to detect the early apoptosis induced by CNHK500-hTRAIL and Ad- hTRAIL. Results CNHK500-hTRAIL could selectively proliferated in SMMC-7721 with an increase of 225137-fold in 96h post-infection. CNHK500-hTRAIL could kill SMMC-7721 cells at a very low MOI（ MOI = 0. 1 ）, and could induce SMMC-7721 cell lines obviousapoptosis, while it had no significant effect on L02. The expressing level of TRAIL in CNHK500-hTRAIL treated SMMC-7721 cell lines was much higher than that in Ad-hTRAIL treated one （almost 10-fold）. The ability of therapeutic effect, transgene expression and apoptosis inducing were stronger than that of replication-defective Ad-hTRAIL. Conclusion Targeted replication-competent adenovirus carrying human TRAIL gene is more effective than that of replication-defective adenovirus carrying human TRAIL gene in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of cancer therapy.