摘要
目的:构建携带受低氧反应元件(HRE)调控的单纯疱疹病毒胸苷激酶(HSV-TK)基因的重组腺病毒载体,探讨该重组腺病毒对体外培养的肝癌细胞HepG2的特异性杀伤活性。方法:采用Ad Easysystem构建携带受HRE调控TK基因表达的腺病毒Ad-HRE-TK,体外感染肝癌细胞系HepG2后分别在正常和低氧条件下培养。分别采用反转录-聚合酶链式扩增(RT-PCR)及蛋白质印迹(Western blotting)技术检5测TKmRNA及蛋白表达情况,并用不同浓度的丙氧鸟苷(GCV)处理后以MTT法检测细胞的增殖情况。结果:RT-PCR及Western blotting结果显示,仅转染重组腺病毒Ad-HRE-TK并在低氧条件下培养的HepG2细胞特异性表达TK基因及蛋白。HepG2细胞感染Ad-HRE-TK后,在低氧培养条件下对GCV的敏感性明显增加,在感染复数(MOI)为100并用50mg/LGCV处理时,则有95%以上HepG2细胞被杀死。而正常氧浓度下培养组,未能观察到GCV的杀伤作用。结论:低氧条件下,HRE可特异性地促进HSV-TK基因的表达,从而诱导GCV的毒性作用。
AIM ： To investigate the in vitro killing effect of adenovirus - mediated herpes simplex virus thymidine kinase gene （HSV -TK） driven by hypoxic response element （HRE） on hepatoma cell line HepG2. METHODS： Recombinant adenoviral vector Ad - HRE - TK was constructed with HSV - TK under the control of HRE using AdEasy system. Then Ad - HRE - TK was transfected into hepatoma cell line HepG2 and the ceils were cultured under normoxic or hypoxic conditions. After treated with GCV for 3 d, the sensitivity to GCV of HepG2 was measured by MTr method. RESULTS ： Over 95% HepG2 cells infected with Ad - HRE - TK cultured under hypoxic condition were killed when the MOI was 100 and the concentration of GCV was 50 mg/L. On the contrary, no killing effect of GCV was observed in cells cultured under normoxic condition. CONCLUSION： HRE promotes the expression of HSV -TK specifically under hypoxic condition and induces the specific killing effect of GCV.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第8期1513-1516,共4页
Chinese Journal of Pathophysiology
基金
广东省科技计划资助项目(No.2005B30301020)
关键词
低氧反应元件
单纯疱疹病毒胸苷激酶
腺病毒载体
肝肿瘤
Hypoxic response element
Herpes simplex virus thymidine kinase
Adenoviral vector
Liver neoplasms
