HSV-tkGCV自杀基因疗法及其影响树突状细胞功能的实验研究

Experimental studies on the characteristics of suicide gene system HSV-tk/GCV and its effect on dendritic cells

目的 研究单纯疱疹病毒 胸苷激酶 更昔洛韦 (HSV tk GCV)自杀基因系统的特性 ,探讨转HSV tk基因后 ,肿瘤细胞被更昔洛韦 (Ganciclovir,GCV)杀伤时的凋亡现象及对树突状细胞功能的影响。方法 以逆转录病毒法将HSV tk基因转入乳腺癌细胞株MCF 7中 ,以Southernblot法对肿瘤细胞基因组DNA中tk基因的整合进行鉴定 ,以流式细胞仪及电镜观察GCV杀伤MCF 7 tk细胞时的凋亡现象。由脐血CD34+ 细胞诱生树突状细胞 ,以3H TdR掺入法检测树突状细胞刺激同种异体T淋巴细胞增殖的能力。结果 以逆转录病毒法成功地将HSV tk基因转入MCF 7细胞中 ,转染tk基因的MCF 7细胞能被GCV有效杀伤并存在凋亡现象 ,凋亡率可达 31.3 % ;由脐血CD34+ 细胞在GM CSF、TNF α、SCF及FL作用下诱生树突状细胞 ,CD1a+ 细胞比例为 (2 7.18± 1.5 6 ) % ,HLA DR表达率为 (93 .7± 1.0 ) % ,电镜下见树突状细胞的典型形态。树突状细胞与凋亡的肿瘤细胞共育后 ,其刺激T细胞增殖的能力增强 (P <0 .0 1)。结论 HSV tk GCV自杀基因系统中 ,肿瘤细胞MCF 7 tk被GCV杀伤时存在凋亡现象 ,树突状细胞的功能可因接触凋亡细胞而改变 ,提示以树突状细胞为基础的肿瘤生物治疗可结合自杀基因系统联合抗瘤。

Objective To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase(HSV tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on dendritic cells(DC). Method HSV tk was transduced into the breast cancer cell line MCF 7 cells with a retrovirus vector. Apoptosis was examined by electro microscopy and FACS. DCs were induced from cord blood CD 34 + cells with a combination of cytokines (GM CSF, TNF α, SCF and FL) and 3H TdR was adopted to determine the proliferation of T cells. Results In vitro experiment showed a dose dependent cell killing of the HSV tk gene transducted cells on ganciclovir(GCV) treatment. The apoptosis rate increased to 31.3% from the control group of 19.3%. Apoptosis is the important mechanism in bystander effect of HSV tk/GCV system. The CD 1a + and HLA DR + cells in the induced DC were of (27.18±1.56)% and (93.7±1.0)%,respectively. Co incubation of DC with apoptotic cells further stimulated the proliferation of T cells. Conclusion It suggested that HSV tk/GCV system, alone or combined with radiotherapy, be a promising suicide gene therapy for cancer patient and an approach to enhancing DC function.