腺病毒载体介导单纯疱疹病毒胸苷激酶基因对雄激素非依赖性前列腺癌细胞的杀伤作用

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy for experimental prostate cancer

目的观察腺病毒载体介导单纯疱疹病毒胸苷激酶（HSV-TK）基因对雄激素非依赖前列腺癌细胞的杀伤作用，探讨雄激素非依赖性前列腺癌（AIPC）基因治疗的可行性。方法制备携带HSV—TK的重组腺病毒载体（AdTK），AIPC细胞PC-3细胞加入AdLacZ，滴度（MOI）分别为50、100、150、200、250。48h后计算各滴度AdLacZ转染PC-3细胞的细胞感染率（％）。转染后使用更昔洛韦（GCV）处理，浓度为10mg／L，噻唑蓝（MTT）比色法观察AdTK／GCV对PC-3细胞的体外杀伤效应。结合电镜技术、流式细胞仪检测探讨AdTK／GCV对PC-3细胞的杀伤机制。结果对体外培养的PC-3细胞杀伤实验表明，随着AdTKMOI增加及GCV剂量增加，细胞存活率逐渐降低。AdTKMOI为150和250时，GCV的半数致死量（IC50）分别为7．75mg／L和5．00mg／L。电镜和流式细胞仪检测证实细胞死亡有细胞凋亡机制参与。AdTK／GCV处理后非转染PC-3细胞出现死亡，存在旁观者效应，效应强弱与AdTK转染、未转染细胞混合比例相关。结论AdTK/GCV能有效杀伤雄激素非依赖前列腺癌PC-3细胞。

Objective To study the killing effects of herpes simplex virus thymidine kinase gene/ ganciclovir （HSV-TK/GCV） system mediated by adenovirus vector on the androgen-independent prostatic cancer （AIPC） cell line PC-3 in ivtro and explore the possibility of gene therapy for AIPC. Methods The plasmid pAdTK containing TK gene and pJM17 with the main Adv gene fragments were co-infected into 293 cells by recombinant DNA technology. PC-3 cells were cultured with AdLacZ. MOI was 50, 100, 150, 200 and 250. After 48 h, the transfection efficacy was calculated. The transfected cells with AdTK were treated with prodrug GCV （ 10 rag/L） in vitro. Killing effects were evaluated by methyl thiazol tetrazolium （MTF） assay. Electron microscopy and flow cytometry were utilized for assessing efficacy of AdTK/GCV system and related killing mechanisms were analyzed. Results The titer of prepared AdTK was 1 × 10^11 pfu/ml. The outcomes indicated survival rate was decreased with the increase in AdTK MOI and GCV concentration. IC50 of GCV was 7. 75 mg/L and 5. 00 mg/L respectively when the AdTK MOI reached 150 and 250. There was significant difference in suicide effects of TK gene. Apoptosis mechanism was demon- strated to be involved in such suicide course as well as bystander effects. Conclusion AdTK/GCV gene therapy may play a key role in inhibition of growth of PC-3 cells in vitro.