【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。更多还原

【Abstract】 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 cells were cultured under normoxic or hypoxic conditions.After treated with GCV for 3 d,the sensitivity to GCV of HepG2 was measured by MTT 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.更多还原