【作者】 于春泳；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 胶质瘤是常见的一种原发于神经系统的恶性肿瘤,是治疗效果最差的肿瘤之一。最大的难点在于复发,这与胶质瘤细胞的无限增殖能力和侵袭性生长有关。近期发现脑胶质瘤中存在胶质瘤干细胞,但仅占细胞成分中的少部分。它具有能自我更新和增殖、分化能力,是形成不同分化程度胶质瘤细胞的“种子”细胞,在胶质瘤的发生、发展和复发中起决定性作用。近年来通过对NOTCH-1信号通路的深入研究,发现NOTCH-1信号的异常与人类肿瘤形成有密切的联系。在多种组织肿瘤中均发现NOTCH-1信号通路的异常激活。中枢神经系统中NOTCH-1信号通路的异常激活可能导致脑肿瘤形成。NOTCH-1信号通路在脑胶质瘤的发生中起重要作用,一些胶质瘤和髓母细胞瘤以及相应的模型系统的维持和存活需要NOTCH-1信号通路信号;NOTCH-1信号通路可能参与了胶质瘤的启动,而且通常作用于神经前体细胞或有神经干细胞特性的细胞。NOTCH信号通路通过NOTCH-HES信号转导系统抑制神经干细胞分化为神经细胞和胶质细胞,从而间接维持神经干细胞的未分化状态和自我更新。在此过程中发挥关键的作用的是碱性螺旋-环-螺旋( basic-helix-loop-helix, bHLH)基因。NOTCH-1信号也可通过细胞周期因子、抗凋亡因子调控,或与其他信号通路如SHH通路协同,发挥调控作用,从而影响肿瘤细胞的增殖和存活。鉴于NOTCH-1信号通路在NSC或神经前体细胞的自我更新、增殖及分化中起重要作用,而在包括髓母细胞瘤在内的胶质瘤和其它多种肿瘤组织及细胞系中也发现NOTCH-1信号通路的异常活化,我们推测在胶质瘤的种子细胞胶质瘤干细胞中应该也有NOTCH-1信号的异常变化,并可能对胶质瘤的发生、发展起重要作用,但是究竟如何变化?这些变化又是通过何种机制决定胶质瘤干细胞的命运及调控其增殖、分化等生物学行为?目前国内外尚未见报导,值得进一步深入研究。在第一部分实验中,采用以CD133为标志,用免疫磁珠法从人脑胶质瘤组织和细胞株中分离脑胶质瘤干细胞并进行体外培养,通过免疫荧光技术检测干细胞标志物CD133、Nestin,诱导分化后检查分化细胞标志物MAP2、GFAP、MBP以及电镜超微结构观察和移植SCID鼠致瘤实验,对其干细胞特性加以鉴定,得到如下结果:不同病理分级的新鲜胶质瘤标本和胶质瘤细胞株中存在一小部分CD133+的胶质瘤细胞,能表达干细胞的标志物CD133和Nestin,符合干细胞的超微结构特点,体外培养能连续传代;具有多向分化潜能:诱导分化后能产生MAP2、β-TubulinⅢ、GFAP、MBP染色阳性的细胞;移植SCID鼠后能形成与亲本肿瘤表型一致的移植瘤。因此,这一小部分CD133+胶质瘤细胞具有干细胞的属性,就是胶质瘤中的肿瘤干细胞,即胶质瘤干细胞。在第二部分实验中,采用体外分离培养的胶质瘤干细胞作为细胞模型,研究胶质瘤干细胞增殖、分化培养过程中NOTCH-1信号通路基因和蛋白的表达情况,以及脑胶质瘤组织和细胞株中的蛋白表达情况及细胞类型,并分析与脑胶质瘤干细胞增殖、分化的关系。应用免疫荧光技术、RT-PCR、Western Blot和流式细胞术等检测对体外培养的脑胶质瘤干细胞增殖、分化、得到如下结果:1、脑胶质瘤组织和细胞株中有NOTCH-1和HES-1表达,而正常成人脑组织中仅能检测到微弱的表达;NOTCH-1和HES-1在高级别胶质瘤组织中的表达强于低级别胶质瘤组织中。2、NOTCH-1和HES-1在CD133、MAP2、GFAP和MBP阳性细胞中均可表达,但在CD133阳性细胞中有很强的表达,而在发育较成熟的MAP2、GFAP和MBP阳性细胞中表达减弱或极弱检测不到,提示NOTCH-1和HES-1的表达强弱可能参与了胶质瘤细胞未分化状态的维持和分化的调控。3、胶质瘤干细胞增殖过程均能检测到较强的NOTCH-1信号通路基因mRNA和蛋白表达,提示NOTCH-1信号通路的强表达可能参与了脑胶质瘤干细胞增殖过程的调控和干细胞状态的维持。4、胶质瘤干细胞分化时NOTCH-1信号通路基因mRNA和蛋白表达逐渐减弱,CD133表达逐渐减弱,MAP2、GFAP和MBP出现表达并逐渐增强,提示NOTCH-1信号通路表达减弱可能参与了胶质瘤干细胞分化的调控。在第三部分实验中,在第二部分的基础上构建了针对人NOTCH-1基因的RNA干扰逆转录病毒载体,应用RNAi技术抑制脑胶质瘤干细胞中NOTCH-1信号通路的表达和作用,分析NOTCH-1信号通路对脑胶质瘤干细胞增殖与分化的调控作用,得到如下结果。1、成功构建了pSiRNA- NOTCH-1和pSiRNA-NC,并经PT 67细胞包装后生成了相应的逆转录病毒载体。2、对NOTCH-1信号通路进行RNAi后,胶质瘤干细胞增殖受到抑制,存活细胞减少;G0/G1期细胞减少,G2/M期细胞增加,凋亡/坏死细胞增加,提示NOTCH-1信号通路对胶质瘤干细胞增殖、细胞分裂和存活有促进作用,该作用可能是通过调控细胞周期以促进细胞分裂、减少细胞凋亡/坏死实现的。3、对NOTCH-1信号通路RNAi后,脑胶质瘤干细胞分化受到抑制,死亡细胞明显增加,说明NOTCH-1信号通路促进胶质瘤干细胞分化和存活。总之,通过本课题的研究,结果表明: NOTCH-1信号通路的基因和蛋白在脑胶质瘤的增殖、分化和存活的过程中同样表达并发挥重要的调控作用,不仅促进脑胶质瘤干细胞的增殖和存活,同样也促进并调控脑胶质瘤干细胞的分化过程和存活,该作用可能是通过调控细胞周期以促进细胞分裂、减少细胞凋亡、坏死实现的。我们也应该看到,NOTCH-1信号仅仅是胶质瘤增殖、分化和存活中起关键作用的信号通路之一,其他的信号通路如、HH、WNT、EGF、bFGF信号通路可能也对胶质瘤增殖、分化和存活起着重要作用,因此,很可能是众多信号通路协同作用,共同完成对胶质瘤干细胞增殖分化和存活的综合调控作用。对这些关键因素的进一步研究,将使我们在更广、更深的层面上认识和理解调控胶质瘤发发病机制。更多还原

【Abstract】 Glioma, as the most frequently malignant brain tumor in central nervous system (CNS), who is the most important problem in treating, due to its unlimited proliferation and invasive growth. Brain glioma stem cells are found in glioma, but they take up a small portion of tumor population,and are capable of self-renew, proliferation and differention. BGSCs are the "seeds" of glioma cells with any differented degree, play a key role in initiation, progression and recurrence of glioma. That is why we choose BGSCs as a cell model.NOTCH-1 signaling pathway was found to be correlated with human tumor formation in recent studies. It was detected in many tumors. Overexpression of NOTCH-1 may give rise to brain tumor in CNS. NOTCH-1 signaling may play a key role in the genesis of brain tumor, especially glioma. NOTCH-1 signaling is essential to maintenance and survival of many gliomas, medulloblastomas and the model system; NOTCH-1 signal may participate in ininitiation of glioma, and usually affect neural precursor cells or neural stem cells. NOTCH-1 signaling take effect through NOTCH-HES, and the expression of ( basic-helix-loop-helix,bHLH) is a reliable gene of cell reaction to the NOTCH-1signal. It may also control proliferation and survival of tumor through Cyclins, anti- apoptosis factor and cooperation with other signals, such as HH signaling.Since NOTCH-1 signaling pathway plays a key role in self-renew, proliferation and differentiation of NSCs or neural precursor cells, and ectopic activation of NOTCH-1 signal was detected in many gliomas (including medulloblastomas), as well as some other tumors and cell lines, we hypothesized that there may be ectopic activation of NOTCH-1 signal in BGSCs, seeds of glioma, which may play a key role in initiation and progression of glioma. But we must kown how did it happen? How did these changes decide the fate of BGSCs and control its proliferation and differentiation?In Part 1 of our research, BGSCs were sorted through immunomagnetic beads marking by CD133 and cultured in vitro, and character as a stem cell was identified by stem cell markers (CD133 and Nestin) and differentiated cell markers MAP2, GFAP and MBP , ultrastructure observing with electron microscope and engrafting to SCID mice for tumorigenesis test.The results were as following:Only a small subset of CD133+ glioma cells in glioma cell lines and fresh specimens from various pathologic grade could express stem cell markers CD133 and Nestin, view ultrastructure of a stem cell and be capacity of serial passage in culture. These CD133+ cells possese a marked capacity for multipotent differentiation and could differentiate into tumor cells expressing MAP2,β-TubulinⅢ, GFAP and MBP; When engrafted into SCID mice, they can generate and form tumors that phenotypically resembl the tumor from the patient. Therefore, the small fraction of CD133+ cells identified stem cells is BGSCs , so called as tumor stem cells in glioma.In Part 2 of the research, BGSCs in vitro were adopted as model. Genes and proteins expression of NOTCH-1 signaling pathway were detected during BGSCs proliferation and differentiation. Proteins expression of NOTCH-1 signaling pathway and cell type were also detected in glioma specimens and cell lines. The relationship between proliferation and differentiation of BGSCs has been analysed. On this basis, a retroviral-mediated expression system containing double strands DNA for RNA interference on human NOTCH-1 gene was constructed. Expression and function of NOTCH-1 signaling pathway in BGSCs were blocked by RNA interference. Immunity fluorescence technique, RT-PCR, Western blot and flow cytometry were used to evaluate effects on proliferation, differentiation, survival and tumorigenesis of BGSCs.The results were as following:1、Expression of NOTCH-1 and HES-1 protein were detected in glioma specimens and cell lines but small in normal adult brain tissue. More intensity of expression were detected in higher grade glioma.2、NOTCH-1 and HES-1 expressed in relativly differentiated and immature cells of CD133,MAP2, GFAP and MBP, especially in the cells of CD133. It implies that expression of NOTCH-1 and HES-1 may be correlated to iniatiation and maintenance of glioma differentiation.3、In proliferating BGSCs, NOTCH-1 mRNA and protein were detected strongly, which implies that the maintenance of strong NOTCH-1 signal may be correlated to stem cell state and survival.4、Expression of NOTCH-1 in differentiating BGSCs increased gradually, then tapered to disappearance; Expression of CD133 tapered too, but the expression of MAP2, GFAP and MBP appeared and increased gradually. Which suggests that proteins expression of may participate in initiation and phase control of BGSCs differentiation.In part 3 of the research, based on the part 2, A retroviral-mediated expression system containing double strands DNA for RNA interference on NOTCH-1 gene was successfully constructed. And by the technique of RNAi, we can found the change of the expression and function of NOTCH-1 signaling pathway. We can analyse the role of NOTCH-1 signaling pathway in BGSCs proliferation and differentiation.The results were as following:1、A retroviral-mediated expression system containing double strands DNA for RNA interference on NOTCH-1 gene was successfully constructed.2、When NOTCH-1 signaling was blocked by RNAi, proliferation of BGSCs were inhibited and viable cells decreased; Cells in G0/G1 phase decreased, in S phase was exhausted or held up, and apoptotic or necrotic cells increased. Which hints that NOTCH-1 signaling could promote proliferation, cell division and survival, and may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis.3、When NOTCH-1 signaling was blocked by RNAi, differentiation of BGSCs were inhibited and dead cells incresed. At the group after RNAi, CD133,β-TubulinⅢ, GFAP and MBP positive cells ratio increased in RNAi group as compared with No-RNAi group and Negative-RNAi group. A inference could be drawn that NOTCH-1 signaling promoted differentiation and survival of BGSCs.It was general believed that the expression of genes and proteins of NOTCH-1 signaling pathway plays a key role in proliferating and differentiating BGSCs, Which promoted not only proliferation and survival but also differentation of BGSCs. It may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis. However, NOTCH-1 signaling is not the only key signal pathways in proliferation, differentiation and survival of BGSCs, some other signals such as HH, WNT, EGF and bFGF may also be at work. It is supposed that numerous signals are involved in controlling the proliferation, differentiation and survival of BGSCs. Further deep and extensive studies on these key factors could show new light on control in that initiation, progression and recurrence of glioma at advanced level.更多还原