摘要目的 探讨内皮抑素和血管生成抑素(Endo-Angio)融合基因修饰的单纯疱疹病毒(VAE)对胶质瘤干细胞(GSCs)荷瘤裸鼠胶质瘤的溶瘤作用.方法 采用人脑胶质瘤标本经原代培养获得稳定传代的GSCs,构建裸小鼠脑内原位GSCs移植瘤模型,瘤内注射VAE治疗,通过MRI观察VAE对GSCs荷瘤裸鼠肿瘤体积的影响,RT-PCR和Western blot检测Endo-Angio融合蛋白的表达,免疫组化CD31染色检测其对微血管密度(MVD)的影响;观察病毒处理后荷瘤裸小鼠的生存情况.结果 (1)从36例胶质母细胞瘤标本中培养出6例稳定传代的GSCs,能够表达CD133,具有自我更新和多向分化能力.(2)VAE感染GSCs荷瘤裸鼠10d后,有Endo-Angio融合基因和蛋白的表达,该融合蛋白使MVD明显降低(p＜0.05).(3)MRI检查发现VAE感染GSCs荷瘤裸鼠后,颅内肿瘤体积明显减小.(4)GSCs荷瘤裸鼠感染VAE后生存时间明显延长(P＜0.05).结论 VAE能感染GSCs荷瘤裸鼠并表达Endo-Angio融合蛋白,杀伤GSCs的同时抑制肿瘤血管生成,为脑胶质瘤病毒-基因治疗开辟了新的途径.

abstractsObjective To explore the effect of endostatin and angiostatin (Endo-Angio) fusion gene modified herpes simplex virus (VAE) on glioma stem cells (GSCs) in vivo.Methods Isolated stable GSC-enriched cultures were obtained from human glioblastoma specimens.After that we established orthotopic nude mice models carrying GSCs.The tumor volumes variation was detected by MRI scan.The expression of Endo-Angio fusion gene at mRNA and protein level was detected by RT-PCR and Western blot.At last,the efficacy of fusion protein to microvessel density (MVD) was detected by immunohistochemistry of CD31 after infected by VAE.Results (1) 6 GSCs isolated from 36 surgical specimens could suspend growth and had the ability of self-renewal and multipotential differentiation,and could express neural stem cells marker,CD133.(2) 10 days after treatment with VAE,the intracranial gliomas derived from GSCs showed significant expression of Endo-Angio fusion gene and reduction in microvessel density (MVD) (P ＜0.05).(3)MRI showed that the tumor volumes of intracranial gliomas generated by GSCs were dramatically reduced at 10 days after treatment with VAE compared with controls.(4) There was a significant improvement in the survival time of mice with intracranial gliomas treated with VAE compared with controls (P ＜ 0.05).Conclusions VAE showed an oncolytic therapeutic efficacy in animal models of human glioblastoma stem cells and expressed endostatin-angiostatin fusion gene,which enhanced antitumor efficacy by reducing the blood supply for tumors.VAE could be used as a novel virus-gene therapy strategy.