TARGET CELL SPECIFIC GENE TRANSFER BY ANTIFECTION AND REGULATION OF PROLIFERATION OF CANCER CELLS

目的：建立一种新的抗体介导的靶向基因转移方法，确定基因转移的最佳条件。材料和方法：OKT3，抗CD3抗体用苯二酚与含有绿色荧光蛋白(GFP)基因的质粒DNA化学偶联。抗体-DNA偶联物通过将混合物分层在40%的蔗糖溶液中，然后进行超速离心法进行浓缩。用1%琼脂糖凝胶电泳法测定DNA与抗体的偶联程度，再用抗鼠IgG抗体进行免疫印迹。将表达CD3的Jurkat T细胞与抗体-DNA偶联物共同孵育，用流式细胞仪检测GFP的表达。结果与讨论：琼脂糖凝胶电泳法显示，与单独的质粒DNA相比，抗体-DNA偶联物的凝胶迁移率发生了变化。免疫印迹显示，抗鼠免疫球蛋白抗体与抗体-DNA反应混合物中观察到的条带发生反应，表明抗体-DNA偶联物的存在。通过将脂类(FuGENE)偶联到抗体-DNA偶联物上，成功地将抗体-DNA偶联物导入Jurkat细胞。氯喹因其细胞毒性而不能成功转导。抗感染可能是一种有用的基因治疗方法。

Purpose : To develop an antifection method, a new technique of antibody-mediated targeted gene transfection, we determined optimal conditions of the gene transfer.Materials and Methods : OKT3, the antibody (Ab) against CD3 was chemically conjugated using benzoquinone to pEGFP, plasmid DNA containing green fluorescent protein (GFP) gene. The Ab-DNA conjugate was enriched by layering the mixture on a 40% sucrose solution followed by an ultracentrifugation. Coupling between DNA and Ab was determined by a 1% agarose gel electrophoresis followed by western blotting by anti-mouse IgG antibody. Jurkat T cells expressing CD3 were incubated with the Ab-DNA conjugate, and then determined the expression of GFP by flowcytometry.Results and Discussion : Agarose gel electrophoresis showed a change of the electrophoretic mobility in the Ab-DNA conjugate, as compared to plasmid DNA alone. The western blot revealed that anti-mouse IgG antibody reacted against the observed band in the Ab-DNA reaction mixture, indicating the existence of the Ab-DNA conjugate. Successful transfection of the Ab-DNA conjugate into Jurkat cells was observed by coupling lipids (FuGENE) to the Ab-DNA conjugate. Chloroquine failed to confer successful transfection due to its cytotoxicity. Antifection could be an useful method for gene therapy.