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

本实验旨在为头颈癌患者开发一种新的基因治疗方法。首先，我们尝试在EB病毒质粒载体（pEBETA3-CAG）中引入自杀基因HSV-tk。当我们用pEBETA 3-CAG-HSV-tk体外电穿孔或脂质体复合物法转染涎腺癌细胞HSG细胞或TYS细胞时，转染的基因在HSG细胞和TYS细胞中均有极高的表达。然而，我们未能将这些ebeta质粒载体导入HSG细胞形成的裸鼠肿瘤中。然后，我们使用含有荧光素酶基因的普通质粒载体pGL3-control作为SV40早期启动子驱动的报告基因，检测了体内转染效率。用pgl3对照转染HSG裸鼠肿瘤，在不同条件下电穿孔。转染2天后，切除小鼠肿瘤，分析组织提取物荧光素酶活性。结果表明，1 kv、99 mb的经皮电刺激可使HSG裸鼠肿瘤转染5 μ g质粒。8倍方脉冲是体内电穿孔最有效的条件。为了检测转染的基因产物在细胞中的定位，我们使用了pEGFP-C3质粒，该质粒含有CMV-IE启动子驱动的绿色荧光蛋白（GFP）。转染2天后，从小鼠身上切除肿瘤，制备冷冻切片。我们在荧光显微镜下观察肿瘤部分细胞中GFP荧光的表达。我们成功地通过电穿孔将质粒载体导入裸体肿瘤。结果表明，在提高转染效率的前提下，该方法可应用于人头颈癌基因治疗。

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.