TK基因联合白介素-2/粒、巨噬细胞集落刺激因子基因治疗胃癌的实验研究

An experimental study on gene therapy of gastric cancer by TK and mIL-2/mGM-CSF genes

目的 用逆转录病毒载体 pLxSN转导TK基因 ,用真核表达载体 pIRES转导细胞因子基因 ,观察TK基因联合白介素 2 (mouseIL 2 ) /粒、巨噬细胞集落刺激因子 (mouseGM CSF)两种细胞因子基因对小鼠胃癌的治疗作用。方法 先用TK基因治疗胃癌细胞株 ,然后联合mIL 2 /mGM CSF基因治疗小鼠模型胃癌 ,其“自杀基因”与细胞因子基因相互协同 ,产生显著的抗肿瘤作用。RT PCR检测基因表达。结果 2 0 %的转基因细胞即可杀灭 70 %～ 80 %以上的肿瘤细胞 ,表现出很强的旁观者效应。实验表明 ,应用TK/ 9 丙氧鸟苷 (GCV ) ,即可产生很强的抗肿瘤作用 (P <0 .0 1)。结合mIL 2/mGM CSF两种细胞因子基因 ,抗肿瘤作用会得到进一步强化 ,肿瘤消退速度明显加快 ,大部分肿瘤完全消退。RT PCR显示 ,TK基因和mIL 2 /mGM CSF基因均能在组织中表达。结论 TK/GCV能够杀灭肿瘤细胞 ,具有显著抗肿瘤作用。联合IL 2 /GM CSF后 ,抗肿瘤作用会得到进一步强化 ,产生明显的抗肿瘤效应。

Objective The TK (thymidine kinase) gene converts GCV to phosphorylated GCV, which inhibits the synthesis of cell DNA. Cytokines also play an important role in anti tumor immunity. IL 2 is a potent activator of T lymphacytes, natural killer cells, and lymphokine activated killer (LAK) cells; GM CSF can potentiate the release of granulocytes and macrophage cells, which transfer the anti tumor message to CD + 4 and CD + 8 T lymphocyte. We combined the TK and mIL 2/mGM CSF genes in order to produce more marked antitumor effect. Methods TK gene was constructed into the retroviral vector pLxSN, the mIL 2 and mGM CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum. The cytokine plasmids caped with liposome were injected into the tumor surrounding tissue. Results In vitro experiment, 20% of TK gene transduced cells could give rise to 70%～80% of total cells to death. In vivo results showed that no treatment effect was noted in control group. TK/GCV could inhibit the tumor growth. The strongest anti tumor effects were shown in TK+mIL 2+mGM CSF group. The pathologic examination showed necrosis in the treated groups.Conclusions TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL 2 and GM CSF could significantly enhance the anti tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues.