【作者】 董志强；

【导师】 陈一戎；

【作者基本信息】 兰州大学 ， 外科学， 2014， 博士

【摘要】 脑胶质瘤是人类中枢神经系统最常见的难治性恶性肿瘤之一,其病因、发病机制、有效治疗方法仍在探索之中。近些年来,人们越来越多地把目光投向胶质瘤的基因治疗。寻找与脑胶质瘤发病相关的基因,深入了解胶质瘤的分子病理,在此基础上寻找胶质瘤治疗的新策略和靶点,成为胶质瘤研究领域的热点问题。p21WAF1/CIP,(p21)基因是细胞周期蛋白依赖性激酶(cyclin dependent kinase, cdk)抑制剂,是目前已知的具有最广泛活性的细胞周期抑制基因,参与了细胞增殖、分化、衰老和凋亡等多种功能的调节。Survivin是近期被发现的凋亡抑制蛋白(inhibitor of apoptosis protein, IAPs)家族成员。主要参与了细胞有丝分裂和凋亡过程。Survivin在肿瘤发生发展、患者预后中的重要作用,使其成为肿瘤综合治疗,尤其是基因治疗的重要靶点。RNA激活(RNA activation, RNAa),是指某些小分子非编码RNA(non-coding RNA, ncRNA)在转录水平激活基因表达的现象。利用RNAa技术特异性激活肿瘤抑癌基因从而治疗肿瘤是肿瘤基因治疗的新思路和新方法。通过RNAa技术特异性上调p21基因的表达,抑制人肾癌,膀胱癌,肺癌,肝癌等肿瘤组织的生长,促进肿瘤细胞凋亡已经得到实验证实,但尚无RNAa在颅内胶质瘤中激活特定的抑癌基因达到治疗肿瘤目的的研究和报道。本课题主要研究人脑胶质瘤细胞中p21和survivin基因的表达与组织学分级之间的关系；以及探讨应用saRNA靶向激活p21对人脑胶质瘤细胞系基因表达,细胞增殖,细胞凋亡和细胞周期分布产生的影响及相关机制,藉此探索临床抗胶质瘤治疗可能的新靶点及新方法。研究内容分为两个部分。第一部分目的：探讨p21及survivin蛋白在人脑胶质瘤细胞中的定位及表达与肿瘤组织学分级之间的关系；方法：应用免疫组化SP法检测Ⅰ-Ⅳ级人脑胶质瘤组织中p21以及survivin蛋白的表达,并分析其与胶质瘤组织学分级之间的关系。结果：p21蛋白阳性染色定位于人脑胶质瘤细胞核中,为棕黄色或棕褐色,不同级别胶质瘤中,p21的阳性表达率不同,胶质瘤级别越高,p21表达水平越低。survivin蛋白阳性染色主要定位于人脑胶质瘤细胞浆内,呈淡黄、棕黄色或棕褐色着色,偶尔可见细胞核着色,不同级别胶质瘤中,survivin的阳性表达率不同,胶质瘤级别越高,survivin表达水平越高。结论：p2l蛋白阳性表达率随着肿瘤病理级别的增高而降低,即恶性程度越高,p2l阳性表达率越低。survivin蛋白阳性表达率随着肿瘤病理级别的增高而增高,即恶性程度越高,survivin阳性表达率越高。第二部分目的：探讨应用saRNA激活p21表达对人脑胶质瘤细胞系基因表达,细胞增殖,凋亡和细胞周期分布的影响。方法：设计抑癌基因p21启动子DNA序列互补的双链RNA分子(dsP21),转染胶质瘤细胞系SHG-44,应用实时荧光定量PCR (RT-PCR)及蛋白质印迹法(Western blotting)检测p21基因、Survivin基因mRNA及蛋白质的表达变化；MTT法检测胶质瘤细胞增殖速度的变化；流式细胞仪检测胶质瘤细胞凋亡率和细胞周期分布的变化。结果：1.dsP21转染胶质瘤细胞72h后,RT-PCR和Western blotting结果显示,p21表达显著上调,survivin表达明显下调,空白对照组、阴性对照组和实验组p21mRNA、蛋白质,Survivin mRNA、蛋白质相对表达量比较差异显著,有统计学意义。2.dsP21转染胶质瘤细胞第三天后,胶质瘤细胞增殖受到明显抑制。3.dsP21转染胶质瘤细胞72h后细胞凋亡率明显增加,细胞周期分布显示G0/G1期细胞明显增多,S期细胞减少。结论：RNAa上调p21基因表达明显抑制了人脑胶质瘤细胞增殖,促进了胶质瘤细胞凋亡,使胶质瘤细胞阻滞在G0/G1期,抑制了人脑胶质瘤细胞survivin基因表达,具有明显的体外抗肿瘤效果；RNAa可以用来特异性激活肿瘤抑制基因治疗人脑胶质瘤。更多还原

【Abstract】 Glioma is the most refractory malignancy in human central nervous system. The cause of disease, pathogenesis and the effective intervention of this tumor are still ambiguous. In recent years, people increasingly pay their attention to gene therapy of glioma. It is become the prevalent issue to pursue pathogenesis related genes, penetrate the molecular pathology and on this basis looking for new strategies and targets to treat glioma.P21WAF1/CIP1(p21) gene is an inhibitor of cyclin-dependent kinase(cdk) and the most widely active gene of cell cycle inhibitor currently known which involved in variety of regulatory functions, such as cell proliferation, differentiation, aging and apoptosis. Survivin was discovered as a IAPs family members recently. Survivin involves in cell mitosis and apoptosis process. Survivin plays an important role in the progression of tumors and the prognosis of patients making it is a significant target for combined modality therapy, especially gene therapy in human’glioma.RNA activation (RNAa), refers to describe the phenomenon of gene activation at the transcriptional level mediated by some small non-coding RNA(non-coding RNA, ncRNA) molecules. This is a new ideas and ways of gene therapies to treating tumors by utilizing RNAa to activate the tumor suppressor gene. It has been confirmed experimentally that the increased expression of p21by specifically activating gene expression may inhibit the growth of human renal carcinoma, bladder cancer, lung cancer, liver cancer and other tumors, but no study has been reported about intracranial glioma.The purpose of this study is to explore the relationship between the expression of p21and survivin in glioma cells and the histological grade of glioma; as well as to investigate the effectiveness and related mechanisms about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21, thereby to discuss the possible new target and new methods for clinical anti-glioma treatment. The study is divided into two parts.The first part Aim:To investigate the expression and localization of p21and survivin protein, and the relationship between the expression of p21and survivin and histological grades in human glioma; Methods:The expression of p21and survivin of I-IV grade human glioma was examined with immunohistochemical technique. The relationship of the expression of p21and survivin with the histological grades of the glioma was analyzed. Results:The positive staining of p21protein positioned in the nuclei of the glioma cells, it was brown or tan. There were significant differences between the positive expression rates of p21and different grade of glioma. With higher malignancy, the expression level decreased. The positive staining of survivin protein positioned in the cytoplasm, occasionally been seen in nuclei of the glioma cells, it was pale yellow, brown, yellow or tan coloring. There were significant differences between the positive expression rates of survivin and different grade of glioma. With higher malignancy, the expression level increased. Conclusions:The decreased expression of p21in glioma was related to the malignancy of the tumor. The increased expression of survivin in glioma was related to the malignancy of the tumor.The second part Aim:to investigate the effectiveness about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21. Methods:Small double-stranded RNA molecules(dsP21) which complementary with p21promoter DNA sequences was designed to transfect glioma cell line SHG-44. Real-time PCR and Western blot analysis were conducted to detect p21and survivin mRNA and protein respectively. Cell proliferation was examined by MTT assay. Apoptosis and cell cycle distribution were detected by flow-cytometric analysis. Results:1. The up-regulated expression of p21mRNA and protein were statistically significant and the down-regulated expression of survivin mRNA and protein were statistically significant in glioma cells compared to blank and negative control groups by detection of real-time PCR and Western blot analysis at72h after transfection with dsP21.2. Since the third days after transfection with dsP21, the proliferation of human glioma cells were significantly depressed.3. The early and late stages of apoptosis were increased in human glioma cells at72h after transfection with dsP21. Analysis of cell cycle distribution revealed that dsP21transfection increased an accumulation in the G0/G1phase and decreased an accumulation in the S phase in human glioma cells. Conclusions:Up-regulation expression of p21by RNAa significantly inhibited cell proliferation, promoted the rates of apoptosis, leaded to G0/G1arrest and remarkably depressed the expression of survivin in human glioma SHG-44cell line. P21activation by RNAa had anti-tumor activity in vitro in human glioma SHG-44cell line. These results suggest that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.更多还原