不同拷贝数CArG/5HRE+HSV-TK系统辅助伽玛刀杀伤C6胶质瘤的初步研究

Cytotoxic effect of gamma knife assisted with different copy numbers of CArG/5HRE+HSV-TK system on C6 glioma cells

目的研究不同拷贝数CArG/5HRE+HSV-TK系统辅助伽玛刀杀伤C6胶质瘤效果,寻求伽玛刀条件下对胶质瘤杀伤更有效的适宜拷贝数CArG/5HRE+HSV-TK系统。方法在Wistar大鼠颅内C6胶质瘤模型的基础上,构建不同拷贝数CArG/5HRE+HSV-TK质粒(空质粒转染+伽玛刀照射组、HSV-TK+伽玛刀照射组、4CArG/5HRE启动子+伽玛刀照射组、9CArG/5HRE启动子+伽玛刀照射组、12CArG/5HRE启动子+伽玛刀照射组),通过脂质体转染到大鼠颅内胶质瘤组织中,对瘤组织伽玛刀放射外科治疗后取大鼠肿瘤组织。通过对瘤组织常规病理、瘤体细胞凋亡、HSV-TK mRNA及蛋白表达的观察,确定不同拷贝数CArG/5HRE+HSV-TK系统辅助伽玛刀诱导HSV-TK表达及对C6胶质瘤杀伤效果。结果各组肿瘤细胞均可见凋亡、破坏,CArG/HRE双启动子+伽马刀照射组更为明显(P<0.05),尤以9CArG/5HRE启动子+伽马刀照射组最为突出,凋亡率为(0.71±0.12)%(P<0.05);CArG/HRE双启动子+伽马刀照射组中HSV-TK mRNA及蛋白表达较无启动子组明显增加(P<0.05),9CArG/5HRE启动子+伽马刀照射组最为明显,HSV-TK mRNA的相对表达量为(0.84±0.09,P<0.05)。结论在SRS治疗条件下,CArG/HRE双启动子能促进目的基因HSV-TK的表达,增加对C6胶质瘤细胞的杀伤,其中,9CArG/5HRE双启动子的作用最明显。

Objective To determine the cytotoxic effect of gamma knife assisted with different copy numbers of CArG/5HRE ＋ herpessimplex virus-thymidine kinase （HSV-TK） system on C6 glioma cells. Methods Intracranial C6 glioma model was established in Wister rats. The recombinant plasmids with different copy numbers of CArG/5HRE and suicide gene HSV-TK were artificially constructed. These different recombinant plasmids were transfected into C6 glioma rats with GCV injected abdominally afterward. After Gamma knife, tumor tissue of rats was collected, and then the killing effect of the tumor was decided by conventional pathology, tumor cell apoptosis and the expression of HSV-TK gene. Results The destruction and apoptosis of tumor cells were observed in all group, and more obvious in the groups containing CArG/HRE promoter （ P 〈 0.05 ）, especially in 9CArG/5HRE group （P 〈 0.05 ）. The mRNA and protein expression of HSV-TK was increased significantly in groups containing CArG/HRE promoter （P 〈 0.05 ） , especially in 9CArG/5HRE group （P 〈 0. 05）. Conclusion Under stereotactic radiosurgery （SRS） treatment, CArG/HRE double promoter significandy promotes the expression of target gene HSV-TK, effect of 9CArG/5HRE double promoter is identified as and enhances the killing effect to C6 glioma cells. The the most significant.