HSV-TK自杀基因对涎腺多形性腺瘤细胞治疗的实验研究

The study of HSV - TK/GCV suicide gene system in the treatment of salivary pleomorphic adenoma cells

目的研究单纯疱疹病毒胸苷激酶基因(Herpes simplex virus thymidine kinase,HSV-TK)/丙氧鸟苷(ganciclovir,GCV)系统对人涎腺多形性腺瘤体外基因治疗的作用。方法采用腺病毒包装重组脂质体介导的含有HSV-TK全长的cDNA真核表达质粒PDC312-HSVTK转染人涎腺多形性腺瘤细胞,通过RT-PCR检测TK基因在转染细胞中的表达;用MTT法检测HSV-TK/GCV系统对多形性腺瘤细胞的杀伤作用和旁观者效应;采用倒置显微镜、组织学染色等观察HSV-TK/GCV系统作用细胞后的形态学改变。结果HSV-TK基因转染24 小时后,肿瘤细胞开始出现空泡性变;转染48小时后,RT-PCR从转染的细胞中成功扩增出1150bp的特异性TK 基因片段。转染36小时后加入不同浓度的GCV治疗,细胞出现核固缩,胞浆裂解,随之细胞死亡、脱壁的现象。细胞存活率随HSV-TK/GCV作用时间延长而逐渐下降,当加入10-4mol/LGCV治疗7天时,HSV-TK/GCV系统的杀伤作用最强,细胞存活率为10.3%。当TK阳性的细胞占细胞总数50%时,其旁观者效应最明显,细胞存活率为31.7%。结论HSV-TK/GCV系统对涎腺多形性腺瘤细胞具有明显的杀伤作用和旁观者效应。

Objective To investigate the effects of herpes simples virus thymidine kinase (HSV- TK)/ganciclovir (GCV) suicide gene system on salivary pleomorphic adenoma (SPA) cells. Methods Expression vector PDC312-HSVTK containing HSV - TK cDNA was transfected into SPA cells. RT - PCR method was used to identify the expressions of TK gene in the transfected cells. MTT method was adopted to check the killing effects of HSV-TK/GCV suicide gene system on the tumor cells. Simultaneously, the observation was performed to assay the morphological alteration of the tumor cells killed by HSV - TK/GCV suicide gene system. Results The tumor cells had a high transfected rate. Twenty-four hours after transfection, some cells showed vacillation in their cytoplasms. Forty-eight hours after transf ection, the special 1150 bp DNA fragment was amplified through RT-PCR from the transfected cells. Thirty- six hours after transfection, different dose of GCV was added, the cells showed nucleus condensation and breakage of cytoplasms, subsequently, the cells went to death and detaching. The survival rate of the cells treated by HSV-TK/GCV was decreased with the days of observation increased. When treated by following 7 days, HSV-TK/GCV suicide gene system showed the most killing effects on SPA cells, the survival rate of the cells was 10. 3 % . When the cells contained TK gene were account for 50 % of total cells, the bystander effect of this system on SPA cells was the most potential. Conclusion HSV-TK/GCV suicide gene system had obvious killing and bystander effects on SPA cells.