Development of a new method of gene therapy for head and neck cancer patients by electroporation of plasmid vector

本实验旨在为头颈部肿瘤患者的基因治疗提供一种新的方法。首先，我们尝试将自杀基因HSV-tk引入EB病毒质粒载体（pEBETA 3-CAG）中。将pEBETA 3-CAG-HSV-tk分别用电穿孔法和脂质体复合法转染人涎腺癌细胞株HSG和TYS，均获得了极高的表达。然而，我们未能将这些EBETA质粒载体导入由HSG细胞形成的裸鼠肿瘤中。然后，我们使用普通质粒载体pGL 3-control检测了体内转染效率，该质粒载体含有SV 40早期启动子驱动的荧光素酶基因作为报告基因。在几种电穿孔条件下，用pGL 3-control转染HSG裸鼠肿瘤。转染后两天，从小鼠中切除肿瘤，并分析组织提取物的荧光素酶活性。结果发现，用5 μ g质粒经皮电击1 μ g，99 μ s转染HSG裸鼠肿瘤。8倍方脉冲是体内电穿孔测试的最有效条件。为了检测转染基因产物在细胞中的定位，我们使用了含有由CMV-IE启动子驱动的绿色荧光蛋白（GFP）的pEGFP-C3质粒。转染后两天，从小鼠中切除肿瘤，并制备冷冻切片。在荧光显微镜下观察到GFP在部分细胞中的表达，并成功地将质粒载体电穿孔导入裸鼠肿瘤中。这些结果表明，该方法可用于人头颈癌的基因治疗，如果转染效率将得到提高。

This experiment was conducted to develop a new method of gene therapy for head and neck cancer patients. First, we attempted to introduce a suicidal gene, HSV-tk, in an EB virus-plasmid vector (pEBETA3-CAG). When we transfected HSG cells or TYS cells, both are salivary gland cancer cells, with pEBETA 3-CAG-HSV-tk in vitro by electroporation or liposome complex method, extremely high expression of the transfected gene was observed in both HSG cells and TYS cells. However, we failed to introduce these EBETA-plasmid vector into the nude mouse tumors formed by HSG cells. Then we examined the in vivo transfection efficiency using a common plasmid vector, pGL3-control containing luciferase gene as a reporter driven by SV40 early promoter. HSG nude mouse tumor was transfected by pGL3-control in several conditions for electroporation. Two days after transfection, tumors were excised from mice, and the tissue extracts were analysed for luciferase activity. It was found that transfection of HSG nude mouse tumors with 5 mug of the plasmid by per-cutaneous electric-shock at 1 kv, 99 musec. for 8 times square pulse is the most effective condition tested for in vivo electroporation. In order to examine the localization of the transfected gene products in the cells, we used pEGFP-C3 plasmid, containing green fluorescent protein (GFP) driven by CMV-IE promoter. Two days after transfection, tumors were excised from mice, and frozen sections were prepared. We obserbed the expression of GFP fluorescent in some of the cells in the tumors under fluorescent microscopy.We succeed to introduce plasmid vector into nude tumors by electroporation. These results suggests that these method can be applied for human head and neck cancer gene-therapy, if the transfection efficiency will be improved.