【作者】 李宾；

【导师】 卢亦成；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 脑胶质瘤是中枢神经系统最常见的恶性肿瘤之一,约占全部脑肿瘤的45-55%。目前,脑胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗和综合治疗等,尽管近年来上述治疗措施已取得长足进步,但由于胶质瘤呈浸润性生长,与周围脑组织无明显分界且易于复发,且疗效均不甚理想。新的治疗手段如基因治疗等虽有望成为治愈胶质瘤的途径之一,但是目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质瘤细胞恶性生物学行为的分子靶标,明确其在胶质瘤发病过程中的病理机制,已成为神经外科领域的研究热点之一。本研究拟通过观察TPX2在不同病理级别脑胶质瘤以及胶质瘤细胞株中的确切表达情况,并通过研究TPX2在胶质瘤细胞株中的异常表达,研究TPX2在胶质瘤发生发展过程中的功能角色。实验共分为三个部分：第一部分,观察TPX2在脑胶质瘤组织中的表达水平以及与胶质瘤病理级别的相关性,调查TPX2的表达水平与胶质瘤患者生存时间的关系；第二部分,观察TPX2在U251和U87脑胶质瘤细胞株中的mRNA和蛋白表达水平,并调查TPX2蛋白在U87细胞内的亚细胞定位；第三部分,通过RNA干扰的方法抑制TPX2基因在胶质瘤细胞内表达,观察其对U87胶质瘤细胞株生长、增殖、凋亡等细胞生物学进程的影响.以及TPX2对部分周期和肿瘤相关性蛋白的影响。因此,探讨TPX2参与脑胶质瘤发生发展以及恶变的分子病理机制,有助于明确胶质瘤的恶性生物学行为及机理,有望为胶质瘤的临床治疗提供新的分子靶标。一人脑胶质瘤中TPX2的表达和临床病理相关性及生存分析目的：研究TPX2在脑胶质瘤组织中表达水平及其与胶质瘤病理级别的相关性,TPX2表达水平同胶质瘤患者生存时间的关系。方法：采用免疫组化染色检测5例正常脑组织和52例不同级别脑胶质瘤中TPX2的表达情况。临床调查胶质瘤患者,利用生存分析检验TPX2表达水平同患者生存时间的相关性。然后用实时定量RT-PCR和Western blot杂交分别检测组织中TPX2蛋白和mRNA在的表达水平。并分析TPX2表达水平同胶质瘤级别的相关性。结果：采用免疫组化法检测了5例正常脑组织及52例人脑胶质瘤石蜡标本中TPX2蛋白的表达情况。结果显示,正常脑胶质细胞TPX2蛋白染色为阴性,绝大部分胶质瘤标本中可见TPX2蛋白的阳性表达。在大部分其它类型的胶质瘤中也可见TPX2蛋白的阳性表达。脑胶质瘤的临床病理级别与TPX2的表达强度与肿瘤恶性程度呈正相关关系(P<0.01)；而性别和年龄则无统计学上的差异(P>0.05)。生存分析表明,TPX2免疫组化染色表达水平同患者生存时间成负相关(p<0.05)。根据TPX2的cDNA序列设计相应引物,应用RT-PCR检测TPX2 mRNA在52例不同级别脑胶质细胞瘤和5例正常脑组织中的表达显示：TPX2 mRNA表达水平在胶质瘤组织中明显上调,与正常脑组织相比差别显著(P<0.01)。并且,TPX2 mRNA水平在高级别胶质瘤组织中的表达水平明显高于低级别胶质瘤,二者相比差异显著(P<0.01)同样,利用Western blot法测定TPX2蛋白在上述胶质瘤组织的表达,结果显示：TPX2蛋白在胶质细胞瘤组织中表达明显上调,与正常脑组织相比差别显著(P<0.01)。TPX2在Ⅲ-Ⅳ级(高恶度)胶质瘤中高表达,而在Ⅰ-Ⅱ级(低恶度)胶质瘤中表达量较低,Ⅲ-Ⅳ级与Ⅰ-Ⅱ级TPX2蛋白的表达量相比较有显著差异(P<0.001)。结论：TPX2在脑胶质细胞瘤可见阳性表达。其表达水平同脑胶质瘤的级别呈明显相关,同胶质瘤患者的生存时间呈负相关。第二部分TPX2在U87、U252胶质母细胞瘤细胞株中的表达目的：研究TPX2在胶质母细胞瘤(GBM)细胞系U87和U251中的mRNA、蛋白表达水平以及TPX2蛋白在U87细胞内的亚细胞定位情况。方法：应用RT-PCR法检测TPX2在GBM细胞系U87和U251中的mRNA表达水平,应用细胞Western blot法研究TPX2在U87和U251细胞系中的蛋白表达水平。应用免疫荧光染色,共聚焦显微镜系统检测TPX2蛋白在U87细胞内的表达及亚细胞定位情况。结果：RT-PCR结果提示TPX2 mRNA表达水平在胶质瘤细胞系U87和U251中明显上调,与正常脑组织相比差别显著(P<0.05)。Western blot法测定TPX2蛋白在U87和U251细胞系中的表达,结果表明：二者TPX2蛋白水平明显高于正常脑组织。免疫荧光染色显示,TPX2蛋白定位于U87细胞核内,并且同细胞核内纺锤体密切相关。结论：TPX2位于细胞核内,且在GBM细胞中其mRNA和蛋白表达水平明显高于正常脑组织。第三部分TPX2异常表达对U87胶质瘤细胞生长、增殖和凋亡的作用以及对部分细胞周期相关性蛋白的影响目的：研究通过RNA干扰抑制TPX2基因后对U87胶质瘤细胞株生长、增殖、凋亡的作用。方法：利用RNA干扰抑制U87细胞株内TPX2的表达。观察细胞生长,3H-TdR同位素渗透法测定细胞增殖。流式细胞仪及Hoechst 33258染色检测细胞凋亡测定细胞凋亡。RT-PCR和Western blot测定TPX2异常表达的细胞株Aurora A、Ran、p53、Cyclin D1、Cyclin B1、c-Myc等mRNA和蛋白表达水平变化。结果：转染TPX2 siRNA可有效导致U87细胞内的TPX2表达。抑制了TPX2表达的细胞株同未转染组相比,其生长、增殖明显降低(p均<0.01)。经流式细胞仪检测,TPX2 siRNA转染后U87的凋亡率较对照组明显增加(p<0.01)。U87细胞内TPX2表达抑制后,Aurora A和Ran cyclin B1 mRNA表达均较对照明显降低,而p53和c-Myc mRNA表达均明显增加,cyclin D1的mRNA表达无显著变化。同样,在Western blot实验中我们发现上诉蛋白表达同样有类似结果。结论：TPX2可促进GBM细胞的生长、增殖、侵袭,并导致肿瘤细胞凋亡减少。敲除TPX2表达可促进肿瘤细胞的生长、增殖、侵袭,并促进肿瘤细胞的早期凋亡。同时,抑制TPX2表达可导致细胞周期相关性蛋白Aurora A和Ran的表达明显下调,而肿瘤和凋亡相关性蛋白p53、c-Myc表达明显增加,细胞周期相关性蛋白cyclin B1的表达也明显降低。由此可见,TPX2促进肿瘤生长、增生的作用机制可能是多方面的。抑制TPX2表达可能作为未来胶质瘤靶向治疗的选择之一。更多还原

【Abstract】 Astrocytoma remains the most common primary neoplasm of the central nervous system and accounts for approximately 45-55% of all brain tumors. It represents a heterogenous group of diseases with different degree of malignancy from relatively indolent pilocytic astrocytomas to highly aggressive glioblastomas. Unfortunately, current therapeutic modalities including surgical resections, chemotherapy, radiotherapy or combinations can not ensure a cure and, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytoma tumorigenesis may offer important targets for treatment strategies.Targeting protein for Xklp2 (TPX2) is a cell cycle-associated human protein encoded by a gene located on human chromosome band 20q11.2. Its expression is tightly cell cycle regulated. Aberrant expression of TPX2 has been found in various malignant tumors. These results suggested that TPX2 plays a role in the oncogenesis of some malignancies. In our previous microarray study, we found expression of TPX2 at a higher level in human astrocytomas more than normal control. However, whether TPX2 expression contributes to glioma development and progression is unknown. In the present study, we sought to determine whether and, if so, how TPX2 regulates the growth of astrocytomas. we investigated expression of TPX2 in different grade human astrocytomas and the related cell lines, and the correlations between TPX2 protein levels and grade of differentiation. Hence, further investigation of the functional role of TPX2 in the carcinogenesis of glioma cells may offer a better understanding of malignant behavior of glioma cells.The aim of this study is to clarify the exact role for TPX2 in the oncogenic process of glioma cells by examining its expression pattern in glioma tissues of different grades and glioma cell lines and knocking down its expression level in glioma cell lines. This study is consisted of three main parts:the first part is to investigate the expression level of TPX2 gene in astrocytomas of different grades and normal brain tissues, correlation of TPX2 expression and patient clinical characteristics was investigated by statistic analysis.the second is to investigate the expression pattern of TPX2 in glioma cell lines and finally, the effects of inhibbited TPX2 expression on tumor proliferation and apoptosis are evaluated in U87 glioma cell. Objective:To investigate the expression level of TPX2 gene in astrocytomas of different grades and normal brain tissues and TPX2 expression status of the tumors and the survival time of the patients.Methods:The expression levels of TPX2 mRNA were evaluated by real-time quantitative PCR, and expression levels of TPX2 protein were assessed in 52 astrocytic tumors of different pathological grades and 5 normal brain controls by using immunohistochemistry and western blot. Further, TPX2 protein level was evaluated by the product-limit estimate of the survival function (Kaplan-Meier method), and differences between the survival functions were assessed with log-rank test and confirmed by the generalized Wilcoxon test..Results:Quantitative real time PCR analysis demonstrated elevated expression levels of TPX2/β-actin in high-grade astrocytomas versus low-grade (p<0.01) or normal brain tissues (p<0.01). Further, TPX2 immunoreactivity was predominantly detected in the nucleus of tumor cells, whereas no positive staining for TPX2 was observed in normal brain tissues. Statistical analysis showed increased TPX2 labelling index in high-grade astrocytomas versus low-grade tumors (p<0.01) or normal controls (p<0.01). Kaplan-Meier survival curves indicated that increased expression of TPX2 was significantly associated with poor overall survival of astrocytoma patients (P=0.000). Conclusion:In summary, we demonstrate thatTPX2 is present in either astrocytomas or normal brain tissues examined, and its expression positively correlates with the degree of malignancy at both RNA and protein levels. TPX2 overexpression was significantly associated with clinical stage and patient survival. Methods:The expression level of TPX2 mRNA and protein were evaluated by real-time quantitative PCR and Western blotting respectively in U87 and U251 glioma cell lines. Subcellular localization of TPX2 in U87 cells identified by immunofluorescent staining.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of TPX2/β-actin in U87 and U25 glioma cells versus normal brain tissues (p<0.01). Western blot analysis showed increased TPX2 expression levels in glioma cells versus normal brain tissues (P<0.05). When the U87 cells were immunofluorescently stained, it was observed that TPX2 protein localized in nucleus and it associates with the mitotic spindle.Conclusion:TPX2 is present in glioma cell lines examined, and its expression levels significantly increase at both mRNA and protein levels versus normal brain tissues.Objective:To investigate the effects of inhibbited TPX2 expression on tumor proliferation and apoptosis of U87glioma cell.Methods:The TPX2 siRNA were transfected into U87 glioma eel by RNA interference. After the inhibition of the endogenous TPX2, the cell proliferative activity was assessed by 3H-TdR incorporation test and cell apoptosis was analyzed by flow cytometry and Hoechst 33258 stainning. The expression levels of Aurora A, Ran, c-Myc, p53, cyclin D1, cyclin B1 Protein were measured by RT-PCR and Western blotting respectively.Results:The growth of inhibbited-TPX2 expressed cells was significantly decreased Cmp was significant lower in inhibbited-TPX2 expressed cell than in control cell by 3H-TdR incorporation test (P=0.025). The data of flow cytometric analysis shown the apoptosis percentage of inhibbtied-TPX2 expressed cell was increased relative to control cell. Similar results were obtained with Hoechst 33258 staining (P<0.05). Levels expression of Aurora A, Ran, c-Myc, p53 and cyclin B1 by Western blot were changed in cells campared with control cells. In inhibbited-TPX2 expressed cell, levels of Aurora A, Ran and cyclin B1 expression were significantly decreased, respectively. Levels of c-Myc and p53 mRNA expression were increased in glioma cell, respectively. However, no significant difference for the expression levels of Cyclin D1 was noted. Conclusion:Overall, our results indicate that inhibbited-TPX2 expression can inhibit cell proliferation and induce apoptosis. Knock-down of TPX2 expression may effect cycle associated-protein.更多还原