逆转录病毒载体介导TK/GCV与CD/5FC双自杀基因治疗肝细胞肝癌的研究

The retroviral-mediated TK/GCV and CD/5FC double suicide gene transfection for the treatment of hepatocellular carcinoma

目的 探讨TK与CD双自杀基因对肝癌细胞的杀伤作用及旁观者效应 ,并在前药用量、杀伤效力及旁观者效应强弱等方面 ,与单自杀基因比较。方法 将TK与CD双自杀基因导入肝细胞肝癌细胞系HCC 990 3,用不同浓度更昔洛韦 (GCV)及 5氟胞嘧啶 (5FC)、不同混育比例及不同混育时间分别作用于细胞 ,观察在这些条件下的杀伤效应及旁观者效应。结果 GCV与 5FC的有效浓度范围分别是大于 10 -2 μg/ml及大于 2mmol/L ,混育 96h杀伤作用明显高于 2 4h。HCC 990 3/TK +CD细胞占 5 %以上 ,即可观察到旁观者效应。结论 以脂质体介导成功将双自杀基因TK与CD感染肝细胞肝癌细胞系HCC 990 3,双自杀基因具有强大杀伤作用 ,较单自杀基因更为明显 ,其前药用量减少 ,降低了药物的毒副作用 ,旁观者效应更为强大。其杀伤作用与前药浓度、混育比例及混育时间呈正相关。

Objective To explore the cell killing and bystander effects of double suicide gene on hepatic cancer cells(HCC). Methods CD and TK were transferred into the hepatocellular carcinoma cell line HCC 9903/TK+CD. Cell lines were treated at varied mixing time, mixing ratio and concentration of GCV and 5FC.Results The efficient concentration of GCV was more than 10 -2 μg/ml while that of 5FC was more than 2 mmol/L.The cell killing efficacy when mixed for 96 h was higher than that of only 24 h. The cell apoptosis was observed through electronic microscopy after using GCV and 5FC. The GIR of mixed cells was higher than that of CD+TK positive cells, which showed that there was a bystander effect when the tumor cells were killed by TK/GCV and CD/5FC. When the ratio of HCC 9903/CD+TK was above 5% or the ratio of HCC 9903/TK was above 10%, the bystander effect could be observed. Conclusions Compared with single suicide gene therapy, the cell killing efficacy of double suicide gene was higher, the bystander effect was stronger, and the concentration of prodrug was lower when the same cytotoxicity was acquired. The cell killing efficacy was positively correlated with the concentration of prodrug, mixture ratio and mixture time.