Sun Xiao-Yi, Wu Zai-De, Hu Jun-Bo
Department of Surgery, Tongji Hospital, Tongji Medical College of Huazhong University of Science & Technology, Wuhan 430030, China.
Hepatobiliary Pancreat Dis Int. 2002 Aug;1(3):373-7.
To study the induction of sensitivity to ganciclovir (GCV) or acyclovir (ACV) in human hepatocellular carcinoma (HCC) cell line transferred by an Epstein-Barr virus (EBV)-based replicon expression vector carrying the herpes simplex virus thymidine kinase (HSV-tk) gene, including killing and "bystander" effect, and also the gene delivery procedure and route of gene therapy in vivo for HCC.
Liposome-entrapped plasmid pDR2/tk was transferred into HCC cells, and then different concentrations of GCV or ACV were added. The transferred cells were mixed with untransferred HCC cells in different proportion and 200 micromol/L GCV was then added into each well. After 72 hours, all samples were measured by MTT colorimetric assay. An EBV-based plasmid eukarotic expression vector carrying IL-2 cDNA was used. Three models of gene direct injection in the local liver, injection through the portal vein, and injection through the embolized hepatic artery were established in closed Wister rats. For each model, two subgroups, injected either naked plasmid DNA or lipofectin-plasmid complex were included. The expression of the IL-2 gene was regularly examined immunohistochemically.
GCV or ACV could apparently kill the transferred HCC cells at a concentration of 0.2 micromol/L. The inhibition rate was changed with different drug concentrations. The "bystander" effect was obviously induced at a transferred to untransferred HCC cells ratio of 1:5. IL-2 gene expression was observed in liver cells of all animals on day 3, which reached peak within 3-7 days, and declined after day 7. Injection of naked plasmid DNA through the hepatic artery plus embolization obtained a best expression.
EBV-based vector is suitable for carrying suicide gene therapy for hepatocellular carcinoma. Gene direct delivery in vivo combined with interventional surgery can be used to treat hepatocellular carcinoma.
研究携带单纯疱疹病毒胸苷激酶（HSV - tk）基因的基于爱泼斯坦 - 巴尔病毒（EBV）的复制子表达载体转染人肝癌（HCC）细胞系后对更昔洛韦（GCV）或阿昔洛韦（ACV）敏感性的诱导情况，包括杀伤和“旁观者”效应，以及肝癌体内基因治疗的基因传递程序和途径。
将脂质体包裹的质粒pDR2/tk转染至肝癌细胞，然后加入不同浓度的GCV或ACV。将转染后的细胞与未转染的肝癌细胞按不同比例混合，随后向各孔中加入200 μmol/L的GCV。72小时后，采用MTT比色法检测所有样本。使用携带白细胞介素 - 2（IL - 2）cDNA的基于EBV的质粒真核表达载体。在封闭的Wistar大鼠中建立三种基因直接注射模型，即局部肝脏注射、门静脉注射和经栓塞肝动脉注射。对于每种模型，包括注射裸质粒DNA或脂质体 - 质粒复合物的两个亚组。定期通过免疫组织化学法检测IL - 2基因的表达。
GCV或ACV在浓度为0.2 μmol/L时能明显杀伤转染后的肝癌细胞。抑制率随药物浓度不同而变化。当转染细胞与未转染细胞比例为1:5时，明显诱导出“旁观者”效应。所有动物肝细胞在第3天观察到IL - 2基因表达，在3 - 7天达到峰值，7天后下降。经肝动脉加栓塞注射裸质粒DNA获得最佳表达。
基于EBV的载体适用于肝癌的自杀基因治疗。体内基因直接传递联合介入手术可用于治疗肝癌。
