电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒，测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高，GFP可在肌细胞内持续高水平表达3周以上，而在肿瘤细胞内只能表达4～6 d，但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感，单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想，但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移，甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.

Target Gene Transfer Mediated by Electroporation for Cancer Therapy in vivo

A plasmid encoding green florescent protein (GFP) was first used to test efficiency of electroporation and optimize parameters for electroporation in vivo. GFP plasmid was efficiently delivered into muscle by electroporation and robust GFP expression in muscle could be observed more than three weeks. While much less GFP positive cells were observed in tumor and GFP expression could only last 6 days but tumors treated with high voltage/short pulse showed about 2.68 fold more GFP positive cells than tumors treated with low voltage/long pulses. The optimized electroporation parameters was used to mediate therapeutic gene transfer into subcutaneous tumors which derived from T739 mice bladder transitional cell carcinoma cell line (BTT-gfp), human mammary carcinoma cell line (MCF-7) and human hepatoma cell line (SMMC 7721-gfp). Those therapeutic genes included immune reaction regulation factors interleukin12, interleukin2 and GM-CSF or anti-angiogenesis factors such as antisense VEGF121cDNA, soluble form of VEGF receptor (sFlk-1) and Tie2 (ExTek). Inhibition of tumor growth and metastasis were observed in T739 mice carried bladder transitional cell carcinoma and nude mice carried either human breast cancer or liver cancer which were treated with multiple transfers of plasmid encoding interleukine12 mediated by electroporation. MCF-7 and SMMC 7721-gfp derived tumor showed sensitive to single anti-angiogenesis gene therapy, yet definite suppression of growth and metastasis of BTT-gfp tumor was resulted from co-tranfer of sFlk-1 and ExTek gene mediated by electroporation. The results suggest that electroporation is a high efficient, safe and economical method for gene transfer in vivo and electro-gene therapy would be a useful method for solid tumor.