非增殖型腺病毒和肿瘤特异性增殖型腺病毒携带IL-12基因治疗肝癌的体外实验研究

目的 比较携带小鼠IL-12基因的增殖型腺病毒(CNHK200-mIL12)和非增殖型腺病毒(Adv-mIL12)对IL-12基因的表达以及对肝癌细胞的杀伤能力。方法 通过MTT以及病毒增殖实验．评估E1B-55000缺陷的增殖型腺病毒CNHK200-mIL12和ONYX-015(dl1520),以及非增殖型腺病毒Adv-mIL12对人正常肝细胞株LO2、人肝癌细胞株HepG2和Hep3B的杀伤能力。采用蛋白质印迹分析和ELISA法,检测CNHK200-mIL12和Adv-mill2感染HepG2和Hep3B细胞后,小鼠IL-12基因的表达情况。结果 CNHK200-mIL12感染HepG2和Hep3B细胞后大量增殖,在感染后96h时检测,分别增殖3160倍和630倍,在极低的MOI(空斑形成单位／细胞)值和极短的时间内(HepG2细胞：MOI=0．2,第4天;Hep3B细胞：MOI=0．005,第2天),可大量杀伤肿瘤细胞,而对LO2细胞无明显杀伤。CNHK200-miLl2和Adv-mIL12感染HepG2细胞后,其IL-12基因表达量,前者是后者的101倍;感染Hep3B细胞后,前者是后者的20倍。结论 增殖型腺病毒载体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。

In vitro gene therapy of hepatocellular carcinoma using replication-defective and tumor-specific replication-competent adenovirus carrying interleukin-12 gene

Objective To evaluate the therapeutic effect and the expression level of a tumor specific replication competent adenovirus and a replication defective adenovirus expression mouse recombinant IL 12 (mIL 12) gene on hepatocellular carcinoma (HCC) in vitro. Methods The cytotoxicity of replication competent adenovirus with E1B 55 000 attenuated CNHK200 mIL12 and ONYX 015 (dl1520), and replication defective adenovirus Adv mIL12 were evaluated by MTT and replication assay in two HCC cell lines (HepG2 and Hep3B) and human normal hepatocyte line (LO2). Western blot and ELISA were used to determine the expression level of mIL 12. Results CNHK200 mIL12 replicated in HepG2 and Hep3B with an increase of 3,160 fold and 630 fold respectively in 96 h post infection. CNHK200 mIL12 could kill HepG2 and Hep3B cells at a very low MOI (Multiplicity of Infection) and in short time course(HepG2:MOI=0.2, on day 4; Hep3B:MOI=0.005, on day 2), while it had no significant effect on LO2. Furthermore, the expressing level of mIL 12 in CNHK200 mIL12 treated HCC cell lines was much higher than that in Adv mIL12 treated one(HepG2 101 fold,Hep3B 20 fold respectively). Conclusion Replication competent adenovirus is more effective than replication defective adenovirus in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of HCC therapy.