Cx43基因提高自杀基因治疗恶性胶质瘤疗效的实验研究

Experimental study on the anti-malignant glioma effect of HSV-tk/GCV suicide gene therapy system enhanced by Cx43 gene

目的探讨连接蛋白43(Cx43)基因联合自杀基因提高恶性胶质瘤治疗疗效的可行性。方法体外实验以脂质体介导Cx43和腺病毒介导HSV-tk基因转染入C6鼠胶质瘤细胞,给以不同浓度的更昔洛韦(GCV),以MTT法检测细胞生存率。体内实验以C6鼠胶质瘤细胞和SD大鼠建立动物模型,原位注射Cx43和HSV-tk基因,腹腔给以GCV,观察荷瘤鼠生存期:强化MRI动态监测荷瘤鼠肿瘤大小:采用原位杂交方法检测Cx43和HSV-tk基因的表达:TUNEL法检测细胞凋亡。结果Cx43基因在体内、外均明显提高HSV-tk/GCV对胶质瘤的疗效。当tk基因转导的细胞约占50%时,Cx43基因联合HSV-tk基因转染C6的GCV ID_(50)和ID_(100)均较单独HSV-tk转染的降低10倍。体内Cx43基因联合HSV-tk/GCV治疗使荷瘤鼠生存期显著延长(P<0.001),凋亡明显增加,其中3只荷瘤鼠的强化MRI示肿瘤于8周消失。结论Cx43基因和HSV-tk基因联合基因治疗可作为临床上胶质瘤联合基因治疗方案的一种优化组合。

Objective To study on the feasibility of enhancing the anti-tumor effect of suicide gene therapy using herpes simplex virus thymidine kinase/gancyclovir （HSV-tk/GCV） system for malignant gliomas by connexin43 （Cx43） gene. Methods C6 rat glioma cells cotransfected in vitro with lipofectamine-mediated Cx43 cDNA and adenovirus-mediated HSV-tk gene were exposed to ganciclovir （GCV） with different concentrations and MMT assay was applied to detect the survival rate of cells. Then the rat models were established by in vivo injection of C6 rat glioma cells into the right caudate nucleus of Sprague-Dawley （SD） rats. Cx43 and HSV-tk genes were injected in situ and GCV was injected intraperitoneally; the survival time of all the tumor-bearing rats were observed; dynamic contrast-enhanced MRI was used to monitor the size of gliomas in tumor-bearing rats; in situ hybridization assay was used to detect the expressions of Cx43 and HSV-tk genes; and, TUNEL assay was used to detect apoptosis ofglioma cells. Results Cx43 evidently improved the anti-tumor effect of HSV-tk/GCV system on gliomas in vivo and in vitro by enhancing the bystander effect when about 50% of C6 glioma cells were transfected with tk gene. The concentration of GCV for ID50 and ID100 of C6 cells cotransfected with Cx43 cDNA and HSV-tk gene was 10 times lower than that transfected with HSV-tk gene alone. In vivo study： rats bearing C6 gliomas treated by Cx43 gene transduction and HSV-tk/GCV system had evidently prolonged survival time （P〈0.001） and higher apoptotic rate compared to those treated by HSV-tk/GCV alone. Contrast-enhanced MRI showed that intracranial tumors of three tumor-bearing rats disappeared within eight weeks. Conclusion Cx43 gene transduction significantly enhances the bystander effect of HSV-tk/GCV gene therapy. The combination of HSV-tk/GCV system with Cx43 gene transduction may be optimal for clinical therapeutic trials of gene therapy for malignant gliomas.