CAJ | 학술논문

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、250.48 h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10 mg/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AdTK MOI增加及GCV剂量增加,细胞存活率逐渐降低.AdTKMOI为150和250时,GCV的半数致死最(IC50)分别为7.75 mg/L和5.00 mg/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.
목적 관찰선병독재체개도단순포진병독흉감격매(HSV-TK)기인대웅격소비의뢰전렬선암세포적살상작용,탐토웅격소비의뢰성전렬선암( AIPC)기인치료적가행성.방법 제비휴대HSV-TK적중조선병독재체(AdTK),AIPC세포PC-3세포가입AdLacZ,적도(MOI)분별위50、100、150、200、250.48 h후계산각적도AdLacZ전염PC-3세포적세포감염솔(％).전염후사용경석락위(GCV)처리,농도위10 mg/L,새서람(MTT)비색법관찰AdTK/GCV대PC-3세포적체외살상효응.결합전경기술、류식세포의검측탐토AdTK/GCV대PC-3세포적살상궤제.결과 대체외배양적PC-3세포살상실험표명,수착AdTK MOI증가급GCV제량증가,세포존활솔축점강저.AdTKMOI위150화250시,GCV적반수치사최(IC50)분별위7.75 mg/L화5.00 mg/L.전경화류식세포의검측증실세포사망유세포조망궤제삼여.AdTK/GCV처리후비전염PC-3세포출현사망,존재방관자효응,효응강약여AdTK전염、미전염세포혼합비례상관.결론 AdTK/GCV능유효살상웅격소비의뢰전렬선암PC-3세포.
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 pJMl7 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 mg/L) in vitro.Killing effects were evaluated by methyl thiazol tetrazolium (MTT) 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 × 1011 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 demonstrated 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 cellsin vitro.