表皮生长因子受体反义RNA联合γ-刀治疗大鼠C6胶质瘤的实验研究

Experimental study on the combined treatment for rat C6 gliomas with EGFR antisense gene therapy and gamma knife radiosurgery

目的研究表皮生长因子受体（EGFR）反义RNA联合γ-刀治疗对鼠C6胶质瘤的生长抑制作用。方法将1×10^6／15mL C6胶质瘤细胞（每只15mL）接种至SD大鼠右侧尾状核头部，建立SD大鼠C6脑胶质瘤模型。6d后进行MRI检查，确定成瘤后随机分为4组（A：对照组；B：EGFR反义RNA基因治疗组；C：γ-刀治疗组；D：EGFR反义RNA＋γ-刀治疗组），每组12只。B、D组在第6、8天进行脂质体介导的反义EGFR质粒治疗。C、D组第10天进行γ-刀放射外科治疗，给予边缘剂量15Gy。A组未作任何治疗。第11天每组处死2只大鼠进行生物学特性检测，包括PCNA（免疫组化法）、凋亡（TUNEL法）；其余10只在γ-刀治疗后1、2、4及8周进行MRI检查，并观察生存期。结果EGFR反义RNA联合γ-刀治疗组较单一治疗组动物生存期显著延长（P〈0．05），肿瘤细胞PCNA阳性率下降、凋亡率增加，MRI检查证实肿瘤生长缓慢。结论γ-刀放射外科联合EGFR反义RNA治疗C6胶质瘤大鼠，具有更高的肿瘤生长抑制率及肿瘤细胞凋亡率，动物生存时间显著延长。

Objective To evaluate the effects of combined therapy with gamma knife radiosurgery and epidermal growth factor receptor （EGFR） antisense RNA （AS-EGFR） for rat C6 giiomas in vivo. Methods Parental C6 cells （1 ×10^6/15 mL） were implanted stereotactically into the right caudate nucleus of SD rats. Rats with well-established cerebral gliomas were identified by MRI 6 d after implantation, then, the rats bearing gliomas were divided into 4 groups （n=12/group）： control group（A） without treatment, AS-EGFR treatment group （B）, gamma knife treatment group （C）, and combined treatment group （gamma knife ＋ AS-EGFR）（D）. At 6, 8 d after implantation, AS-EGFR treatment was performed, and at 10 d the gamma knife treatment was given at the 50% isodose line （15 Gy）. Group A did not undergo any theatment. At 11 d, 2 rats in each group were executed for biological examination including immunohistochemical staining for the expressions of EGFR and PCNA and TUNEL method for the apoptosis in glioma cells, and the other 10 in each group underwent the dynamic MRI examination at week 1, 2, 4 and 8 after gamma knife treatment. Results Compared with the mean survival time in the rats of control group [（17.5±0.71） d], that in the rats of AS-EGFR group or gamma knife group was prolonged; the survival time of the rats in combined treatment group was significantly prolonged （P〈0.05 vs AS-EGFR group or gamma knife group） and 7 rats kept alive. In the combined treatment group as compared with AS-EGFR group or gamma knife group, the positive rate of PCNA was decrease, the apoptosis rate was increased, and MRI demonstrated that the gliomas grew more slowly. Conclusion The combined treatment of EGFR antisense gene therapy and gamma knife mdiosurgery was more effective than the treatment with either of them in suppressing the tumor growth and inducing the tumor cell apoptosis and prolonging the survival time of rats with C6 gliomas.