靶向Survivin基因的短发卡RNA在体内对U251细胞移植瘤的影响

Impact on U251 Cell Xenograft in vivo through Short Hairpin RNA Targeting Survivin Gene

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 family of inhibitor of apoptosis protein（IAP）,having multiple biologie activities inelnding antiapoptosis.promoting cell proliferation and angiogenesis.however,its role in tumorigenesis and progression of brain glioma is not completely clear.The current study was designed to observe the impact of stable transfection of specific short hairpin RNA（shRNA） eukaryotie expreesion vector targeting survivin gene on tumorigenesis and angiogenesis of human brain gliblastoma U251 cells in nude mice in vivo.METHODS：U251 cells,U251-SR cells transfected stably with shRNA eukaryotie expression vector pWH1-SR targeting survivin gene,and U251-P cells transfected stably with blank pWH1 vector.were inoculated respectively into subcutaneous tissue in flank of nude mice to eslablish xenograft models of human brain glioma.The tumor growth status was observed and tumor volnme was measured termly,and the tumor growth curve was drawn.The animals were killed and the tumor weight was investigated 45 days after inoculation.Protein expression of survivin,proliferating cell nuclear antigen（PCNA）and factor Ⅷ related antigen（F／ⅧRAg）was investigated by inmunohistochemistry SABC method,apoptotie cells were sereened by TUNEL method.furthermore proliferative index （PI）,apoptotic index（AI） and microvessel density（MVD）were measured respectively in each group of tumor specimens.RESULTS：Comparing with those in U251 and U251-P groups.in U251-SR group.the tumorigenesis time delayed.tumor grew slow,both tumor volume and tumor weight deereased significantly（P〈0.01 for both）.surivin protein expression was downregulated markedly,PI and MVD decreased signifieantly,whereas AI in creased remarkably（P〈0.01 for all）.CONCLUSION：The speeifie shRNA targeting survivn gene can inhibit signifieantly tumorigenesis and angiogenesis of U251 cells in vivo.For this survivn gene may act as a valuable target for gene the rapy of glioma,meanwhile,RNA interference（RNAi）technology may provide a novel aand important means for gene therapy of glioma.