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病毒载体(pEBETA3-CAG)中引入自杀基因HSV-tk。当我们通过电穿孔或脂质体复合体的方法将Pebeta3-CAG-HSV-tk导入HSG细胞或TYS细胞时，均可观察到HSG细胞和TYS细胞中都有极高表达的基因。然而，我们未能将这些EBETA载体导入HSG细胞所形成的裸鼠肿瘤中。然后以含有荧光素酶基因的普通质粒载体pGL3-Control为报告载体，检测SV40早期启动子驱动的体内转染率。以pGL3为对照，在不同的电穿孔条件下，将其导入HSG裸鼠移植瘤。在转基因两天后，从小鼠身上切除肿瘤，并对组织提取物进行荧光素酶活性分析。结果表明，在1KV、99musec的条件下，经皮电击可将5杯重组质粒导入HSG裸鼠移植瘤中。8次方波脉冲是体内电穿孔最有效的条件。为了检测基因产物在细胞中的定位，我们使用了含有CMV-IE启动子驱动的绿色荧光蛋白(GFP)的pEGFP-C3载体。两天后，摘除小鼠肿瘤，制备冰冻切片。我们在荧光显微镜下观察到GFP荧光在部分肿瘤细胞中的表达，并成功地通过电穿孔将载体导入裸鼠肿瘤中。这些结果表明，如果能提高转染率，这些方法可以应用于头颈部肿瘤的基因治疗。

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.