摘要
目的观察人端粒酶反转录酶-胸苷激酶/丙氧鸟苷(hTERT-TK/GCV)联合hTERT-胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)对肝癌细胞株HepG2生长及凋亡的靶向杀伤效应。方法细胞培养,构建pGL3-hTERT-CD质粒,hTERT-TK/GCV联合hTERT-CD/5-FC同时转染HepG2细胞和正常肝细胞L-02,用噻唑蓝(MTT)法观察药物浓度对肝癌细胞存活率的影响,用DNA缺口末端标记法(TUNEL)观察自杀基因对肝癌细胞生长和凋亡的影响,以及对旁观者效应的观察。结果MTT法显示hTERT启动子驱动下两自杀基因体系可以协同表达于肝癌细胞,当GCV为20μg/ml,5-FC为200μg/ml时,对HepG2细胞的杀伤作用达到最大。TUNEL法显示在肝癌细胞中,自杀基因一被启动子高效表达,产生凋亡小体和明显的细胞凋亡率,转染正常肝细胞无此现象。结论hTERT-TK/GCV联合hTERT-CD/5-FC基因体系能够靶向杀伤肝癌细胞,存在明显的旁观者效应,弥补了基因治疗转染效率不高的问题,便于指导今后的实验和研究。
Objective To observe the growth and apoptosis of hepatic cell carcinoma （HCC） cell line HepG2 affected by targeted killing of hTERT-TK/GCV combined with hTERT-CD/5-FC. Methods HCC cell line HepG2 and normal hepatic cell L-02 was cultured in vitro. Plasmids of pGL3-hTERT-CD were constructed for transfection of HepG2 and normal hepatic cell L-02 using hTERT-TK/GCV combined with hTERT-CD/5-FC. Survival of HCC cells related to different levels of agents was assessed with MTr method. The bystander effect and growth and aoptosis of HCC affected by suicide gene were studied with TUNEL. Results MTr showed concurrent expression of two suicide genes in HepG2 at the presence of hTERT promoter. The most eminent killing of HepG2 was achieved with 20 μg/ml GCV and 200 μg/ml 5-FC. TUNEL study showed that in HCC but not in normal hepatic cells, suicide gene was effectively expressed by promoter of hTERT, which resulted in apoptotic bodies and a significant rate of apoptosis. Conclu- sion hTERT-TK/GCV combined with hTERT- CD/5-FC led to targeted killing of HCC cells with obvious bystander effect, which can compensate for the low transfecting efficiency of gene therapy, and may have a role in guiding future experiment and researches.
出处
《中国药物与临床》
CAS
2009年第2期91-95,161,共6页
Chinese Remedies & Clinics
基金
国家自然科学基金(30672405)
