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背景与目的:Survivin基因属于凋亡蛋白抑制因子(inhibitorofapoptosis,IAP)家族,具有抗细胞凋亡、促细胞增殖和促血管形成等多种生物学活性,其在脑胶质瘤发生和发展过程中的作用尚不完全明确。本研究旨在观察稳定转染靶向Survivin基因的特异性短发卡RNA(shRNA)真核表达载体对人脑胶质母细胞瘤U251细胞裸鼠体内肿瘤生长和血管形成的影响。方法:将U251细胞,稳定转染Survivin基因shRNA真核表达载体pWH1-SR的U251-SR细胞,以及稳定转染空载体pWH1的U251-P细胞,分别接种于裸鼠背部皮下,建立人脑胶质瘤裸鼠皮下移植瘤模型。定期观察肿瘤生长情况,测定肿瘤体积,绘制肿瘤生长曲线,观察45天后处死动物,称瘤重。采用免疫组化SABC法检测Survivin、增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)以及八因子相关抗原(factorⅧrelatedantigen,FⅧRAg)在各组肿瘤标本中的表达,采用TUNEL法检测凋亡细胞,分别计算各组肿瘤标本的增殖指数(proliferativeindex,PI)、凋亡指数(apoptoticindex,AI)以及微血管密度(microvesseldensity,MVD)。结果:与U251、U251-P组裸鼠相比,U251-SR组裸鼠肿瘤形成时间延迟,肿瘤生长缓慢,肿瘤体积及瘤重均明显减小(P<0.01);U251-SR组肿瘤标本Survivin蛋白表达明显下调;PI和MVD明显减少,AI明显升高(P<0.01)。结论:靶向Survivin基因的shRNA能够在体内明显抑制U251细胞的肿瘤生长和血管形成,Survivin基因可作为脑胶质瘤基因治疗的一个良好靶点,而RNA干涉(RNAinterference,RNAi)技术可为脑胶质瘤的基因治疗提供新的重要手段。
BACKGROUND & OBJECTIVE: Survivin gene belongs to the inhibitor ofapoptosis (IAP) family and possesses a variety of biological activities such as anti-apoptosis, pro-proliferation and angiogenesis. During the development and progression of glioma The role is not yet fully understood. The purpose of this study was to investigate the effect of shRNA-specific eukaryotic expression vector targeting Survivin gene on tumor growth and angiogenesis in nude mice bearing human glioblastoma U251 cells. Methods: U251 cells, U251-SR cells stably transfected with Survivin gene shRNA eukaryotic expression vector pWH1-SR and U251-P cells stably transfected with empty vector pWH1 were inoculated into the back of nude mice subcutaneously to establish human brain gel Subcutaneous xenograft tumor model in nude mice. Regular observation of tumor growth, tumor volume determination, mapping of tumor growth, observed 45 days after the animals were sacrificed, said tumor weight. Immunohistochemical SABC method was used to detect the expression of Survivin, proliferating cell nuclear antigen (PCNA) and factor VIII related antigen (FⅧRAg) in each group of tumor samples. The apoptotic cells were detected by TUNEL method and the tumor samples Proliferative index (PI), apoptotic index (AI) and microvessel density (MVD) were measured. Results: Compared with the U251-U251-P group, the tumor formation time of U251-SR group was slow, the tumor growth was slow, the tumor volume and tumor weight were significantly decreased (P <0.01) Survivin protein expression was significantly down-regulated; PI and MVD were significantly reduced, AI was significantly increased (P <0.01). CONCLUSIONS: Survivin targeting shRNA can significantly inhibit tumor growth and angiogenesis in U251 cells in vivo. Survivin gene may serve as a good target for gene therapy of glioma. RNA interference (RNAi) Gene therapy of gliomas offers new and important tools.