AFP启动子与胸苷激酶基因治疗肝癌的实验研究

目的：探讨AFP启动子与胸苷激酶疗法对肝癌的作用。方法：采用含tk基因与潮霉素磷酸转移酶hy基因融合基因的真核表达载体及仅含潮霉素磷酸转移酶hy的空载体，以脂质体为介导，将这两种质粒分别转染肝癌细胞系HEPG2。潮霉素B药物敏感实验筛选HEPG2，不同浓度丙氧鸟苷分别作用于HEPG2，HEPG2-AFP—hytk及HEPG2-hy。结果：潮霉素B药物敏感实验筛选HEPG2，12天时HEPG2全部死亡的最低浓度为60μg／ml，30μmol／L丙氧鸟苷可最大限度杀死HEPG2-AFP—hytk。杀伤效应随时间而增强，作用24小时后开始出现毒性作用，至96小时死亡率〉80％。仅含有18％的HEPG2-AFP—hytk就可使周围20％的HEPG2细胞死亡，总死亡率为38％。结论：60μg／ml潮霉素是筛选HEPG2－AFP—hytk及HEPG2－hy阳性克隆的最佳剂量。30μmol／L丙氧鸟苷是作用于HEPG2－AFP—hytk的最佳剂量。HSV—tk／GCV系统大于18％HEPG2-AFP—hytk开始具有旁观者效应，可提高疗效。

Experimental study on HSV- tk gene therapy of hepatocellular carcinoma

Objective：To study the effect of HSV - tk suicide gene therapy for hepatocellular carcinoma. Methotis： A vector LXpsp - AFP - hytk which contains hy - tk fusion gene and a vector LXSH which contains only hygromycin phosphotransferase （hph） gene were used in the experiments. These two vectors were transduced separately into two groups of hepatocellular carcinoma cell line, HEPG2 ,by lipofectin. HEPG2 was selected by hygromycin B. The dosage of hygromycin B was determined as following：all cells of HEPG2 died after twelve days,the lowest concentration of hygromycin B was 60μg/ml. Results ：60μg/ml hygromycin B was the best dosage to select HEPG2. The hy - tk cell clone and hy cell clone were obtained by hygromycin B at the dosage of 60μg/ml in this experiment, and named as HEPG2 - AFP - hytk and HEPG2 - hy respectively. There was no difference in the growth curve of HEPG2, HEPG2 -hy and HEPG2 -AFP- hytk. Different Ganeiclovir （GCV）dosage was given to HEPG2, HEPG2 -hy and HEPG2 - AFP - hytk. The cytotoxic effect of GCV on HEPG2 - hy - tk cells （0.03 - 400μmol/L） was obvious （ IC50 =0.03μmol/L） ,while GCV almost showed no any cytotoxic effect on HEPG2 and HEPG2 -hy cells （IC50 〉 400μmol/L）. HEPG2 -AFP- hytk was killed by 30μmol/L GCV effectively. Over dosage of GCV didn＇t show any adding effect. The killing effect of 30μmol/L GCV on HEPG2 -AFP -hytk cells strengthened as time went on, killing began after 24 hours,and killed over 80% of cells after 96 hours. At the dosage of 30μmol/L GCV for 120 hours, 38% of the cells of HEPG2 - AFP - hytk and HEPG2 - hy mixture were killed when HEPG2 - AFP - hytk occupied only 18% of the mixture culture cells. It obviously showed the bystander effect. Conclusions：The best dosage of hygromycin is 60μg/ml for selecting HEPG2 - AFP - hytk. The best dosage of GCV is 30μmol/L to kill HEPG2 - AFP - hytk. HEPG2 - AFP - hytk has the bystander effect,if it is greater than 18% of the mixture cells of HEPG2 - AFP -hytk in the HSV -tk/GCV system.