双调控溶瘤腺病毒治疗肝癌的研究

The antitumor effects of dual - regulated oncolytic adenovirus on hepatocellular carcinoma

目的观察人端粒酶反转录酶（hTERT）与杂合启动子HREAF双调控并携带靶向干扰黏着斑激酶（FAK）短发夹RNA（FAKshRNA）的溶瘤腺病毒SG505-siFAK在体外和体内对肝癌的抗癌作用。方法利用半数细胞培养物感染量（TCIDSO）法检测重组溶瘤腺病毒SG505、SG505．增强型绿色荧光蛋白（EGFP）和SG505-siFAK病毒在不同细胞中的扩增倍数。利用Westernblot检测FAK、早期蛋白E1A和早期蛋白E1B的表达强度。建立裸鼠移植瘤模型，给予溶瘤腺病毒治疗，观察比较肿瘤生长体积，分析FAK免疫组织化学结果。结果TCID50法检测病毒扩增结果显示：SG505-siFAK在Hep3B和HepG2细胞中的扩增倍数为1．36×10^8倍和2．02×10^8倍，明显强于在SMMC-7721、BJ、L02、PANC-1和H460细胞中的扩增能力。SG505-siFAK携带的靶基因FAK．shRNA可以有效降低FAK的表达。Westernblot结果显示SG505、SG505-EGFP和SG505-siFAK感染BJ、L02后E1A、E1B表达较弱，而感染Hep3B、HepG2后E1A、E1B表达较强。噻唑蓝（MTT）法检测细胞增殖结果显示，SG505-siFAK感染Hep3B、HepG2、SMMC-7721、L02、PANC．1和H460的半数抑制浓度（IC50）分另4是（0．092±0．009）、（0．424±0．414）、（14．790±2．520）、（48．709±0．927）、（5．970±0．945）和（2．710±0．244）pfu／cell。裸鼠移植瘤实验结果显示：静脉注射和瘤内注射Wad5、SG505和SG505-siFAK的产生的抑瘤率分别是80．06％、54．14％、79．63％和84．54％、56．64％、87．14％，瘤内注射SG505-siFAK较静脉注射产生更高的抑瘤率（P〈0．01）。免疫组织化学结果显示SG505-siFAK可以有效降低FAK在肿瘤组织的表达。结论SG505-siFAK能选择性地在甲胎蛋白（AFP）阳性肝癌细胞内扩增并产生溶瘤作用，腺病毒SG505自身的溶瘤作用和FAK-shRNA的抑瘤作用表现出协同抗肿瘤效应。

Objective To study the antitumor effects on the liver cancer cells of the novel oncolyt- ic adenovirus SG505 - short - hairpin RNA targeting the focal adhesion kinase （shFAK） , a recombinant adenovirus with the E1A gene driven by human telomerase transcriptase （hTERT） promoter and E1 B gene driven by hybrid HREAF promoter and carrying shFAK. Methods The amplifications of the viruses were measured by the 50% tissue culture infective dose （TCIDS0） assay and the cell viabilities were analyzed by methyl thiazol tetrazolium （MTT） assay. The expression of FAK, E1A and E1B were analyzed by the Western blotting. The nude mice bearing hepatocellular carcinoma were established and treated by various oncolytie adenoviruses. The volumes of the tumors were observed once every week and the FAK expressions in the tumor tissue were examined by immunohistochemistry. Results TCID50 assay revealed the replica- tions of SG505 - siFAK in Hep3B and HepG2 were 1.36× 10^8 and 2. 02 × 10^8 fold which were more potent than those in the SMMC - 7721, BJ, 1332, PANC - 1, and H460. The targeted gene FAK - shRNA armed in the SG505 -siFAK could down -regulate FAK expression in the Hep3B and HepG2. E1A and El B ex- pression determined by the Western blotting were weak in the BJ and L02 infected by the SG505, SG505 - EGFP and SG505 - siFAK and E1A and E1B expression were strong in the Hep3B and HepG2 infected by the SG505, SG505 - EGFP and SG505 - siFAK. The 50% inhibitory dose （ IC50 ） values （ resulting in 50% of cell growth inhibition rate ） of SG505 - siFAK were （ 0. 092±0. 009 ） , （ 0. 424 ± 0. 414 ） , （14.790±2.520）, （48.709 ±0.927）, （5.970 ±0.945）, and （2.710±0.244） pfu/cell in the Hep3B, HepG2, SMMC -7721, L02, PANC - 1, and H460 cells, respectively. In the xenograft tumor model, the tumor inhibition rates of WadS, SG505, and SG505 - siFAK by intravenous injection and intra-tumoral injection were 80. 06%, 54. 14%, 79. 63% and 84. 54%, 56. 64%, 87. 14%, respectively. Tumors treated with SG505 - siFAK by intratumoral injection produced higher tumor inhibition rates compa- ring to the intravenous injection （P 〈 0. 01 ）. The immunohistoehemistry results revealed that the SG505 - siFAK could significantly down -regulate the FAK expression in both groups. Conclusion SG505 - siFAK was a novel dual - regulated oncolytic adenovirus and showed selective replication capability and an- titumor effects in the AFP - positive liver cancer cells and lysed the tumor ceils with minimum adverse effects to the normal cell. The oncolytic effects of SG505 and tumor inhibition effects of FAK - shRNA were comprehensive and synergetic.