【作者】 范左栩；

【导师】 詹仁雅；

【作者基本信息】 浙江大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 研究背景胶质瘤是中枢神经系统最常见的原发恶性肿瘤,因其具有高增殖和侵袭性行为,病情进展迅速,预后差。胶质瘤的治疗一般是以手术切除为主,再辅以放疗和化疗等综合治疗,虽然近年来各种治疗新技术不断出现,但胶质瘤患者的总体预后仍未得到明显改善。针对胶质瘤增殖和存活的分子机制方面的研究突破有助于针对胶质瘤进行更有效的靶向治疗。近年来的研究强调了恶性胶质瘤中肿瘤起始细胞的重要性,这部分细胞被称为胶质瘤干细胞(glioma stem cells, GSCs),具有与大脑神经干细胞相似的生物学特性。它们在胶质瘤细胞中仅占一小部分,但是能自我更新、增殖、多潜能分化并成为与母本形态和分子表型相同的胶质瘤细胞,在胶质瘤的发生发展、侵袭、复发等过程中起着十分重要的作用,是今后胶质瘤治疗的潜在靶向目标。低氧微环境是实体肿瘤的重要特征,对肿瘤的发生发展起着重要的作用。低氧使得肿瘤对放化疗的抵抗性增加,而且能增强肿瘤的侵袭性和不良预后。其中低氧诱导因子-1α(hypoxia-inducible factors-1α, HIF-1α)是启动这一系列生物学改变的一个重要因子,在细胞应对低氧微环境的生理及病理过程中起着关键作用。Notch信号通路参与细胞的生长、分化、增殖,特别是神经干细胞的维持等生物学进程,异常的Notch通路调节与肿瘤发生发展密切相关。有研究表明低氧能够通过激活Notch-1信号通路共同参与肿瘤的发生发展,因此我们有理由推测缺氧及HIF-1α可能通过Notch-1信号通路的激活对胶质瘤干细胞的增殖产生影响。目的(1)检测常氧及低氧环境下培养的胶质瘤细胞球中CD133+细胞比例、细胞活力及HIF-1α表达的变化,探讨低氧微环境对胶质瘤干细胞增殖的影响。(2)检测低氧环境下培养的胶质瘤细胞球和HIF-1α基因敲除细胞球中CD133+细胞比例、细胞活力变化,验证HIF-1α基因是否在低氧环境下培养的胶质瘤干细胞的增殖过程中发挥重要作用。同时通过检测Notch-1通路相关基因的转录和表达水平,来分析缺氧微环境,特别是HIF-1α是否通过激活Notch-1信号通路,从而与之协同表达进而影响胶质瘤干细胞的增殖。方法(1)常氧及低氧培养胶质瘤细胞球24h.48h后,流式细胞术检测CD133+细胞比例,CCK8法检测细胞活力,Real Time-qPCR检测HIF-1α mRNA水平,Western Blot检测HIF-1α蛋白表达水平。(2)利用siRNA建立HIF-1α敲除胶质瘤细胞球；低氧培养两种胶质瘤细胞球24h、48h后,流式细胞术检测CD133+细胞比例,CCK8法检测细胞活力。常氧下培养胶质瘤细胞球24h,低氧培养胶质瘤细胞球及HIF-1α敲除胶质瘤细胞球24h后,流式细胞仪检测细胞周期,Real Time-qPCR及Western Blot检测Notch-1通路相关基因的转录和表达水平。结果(1)相对于常氧环境,低氧环境培养胶质瘤细胞球24h、48h后,CD133+细胞比例、细胞活力和HIF-1α蛋白表达均明显上升；HIF-1α mRNA表达无明显改变。(2)相对于对照组,低氧环境培养HIF-1α敲除胶质瘤细胞球24h、48h后,CD133+细胞比例、细胞活力均明显下降。(3)流式细胞仪检测细胞周期显示,相对于常氧细胞组,缺氧组细胞球G1期细胞比例明显下降,S期及G2期细胞比例明显升高；相对缺氧组,HIF-1β基因敲除组细胞G1期细胞比例明显下降,G2期细胞比例明显升高。(4)缺氧培养胶质瘤细胞球Notch-1通路基因(Notch-1,jaggad-1,HES-1)mRNA及蛋白表达较常氧培养组明显升高,HIF-1α基因敲除组Notch-1通路基因mRNA及蛋白表达较未敲除组组明显降低。结论(1)低氧微环境可以促进胶质瘤干细胞增殖和自我更新,提高胶质瘤干细胞中HIF-lα蛋白的表达,而HIF-1α的mRNA表达水平没有明显改变；通过siRNA干扰HIF-1α基因,可以显著降低低氧微环境中胶质瘤干细胞的增殖和细胞活力。提示HIF-1α基因可能在该过程中发挥关键作用。(2)低氧微环境可以促使胶质瘤干细胞突破G1/S阻滞点,进入S期,从而促进其增殖；而HIF-1α基因敲除以后,可能造成胶质瘤干细胞G2/M期阻滞,从而抑制细胞增殖。(3)缺氧微环境及其核心调控因子HIF-1α可以通过激活Notch-1信号通路来促进胶质瘤干细胞的增殖,HIF-1α基因敲除以后,Notch-1通路受到抑制,从而抑制胶质瘤干细胞的增殖。更多还原

【Abstract】 Background:Gliomas are the most common primary brain tumors and confer a grave prognosis. Standard therapies, such as chemotherapy, surgery, and radiation, have had limited success in treating patients with high-grade gliomas. Despite the continuous development of new clinical therapies, the prognosis and survival of glioma patients remain dismal. It is hoped that a greater understanding of the molecular pathways involved in glioma cell proliferation and survival will lead to more effective targeted therapies.Recent researches highlight the importance of cancer-initiating cells in the malignancy of gliomas. These cells have been referred to as glioma stem cells, as they share similarities to normal neural stem cells in the brain. There is increasing evidence that malignant gliomas arise from and contain these minority tumor cells with stem cell-like properties. This subpopulation of tumor cells with the potential for self-renewal and multi-lineage differentiation that recapitulates the phenotype of the original glioma, plays an important role in glioma initiation, growth, and recurrence. Glioma stem cells seem the potential target of glioma therapy in future.Hypoxic regions are common in malignant tumors such as glioma, and are thought to play an important role in several facets of tumor pathobiology. Hypoxia has been associated with resistance to radiation and chemotherapies in tomor, as well as tumor invasion and poor patient survival. One well-characterized mechanism of the cellular response to reduced oxygen availability involves the enhanced expression of the hypoxia-inducible factor-la (HIF-1α). The Notch pathway, a highly conserved signaling network, is critical for a series of processes, including cell fate specification, differentiation, proliferation, and survival, and especially maintenance of neural stem cells. Dysregulated Notch signaling has been implicated in tumorigenesis.Objectives:(1) By testing the CD133+phenotype, cell vitality and gene expression of HIF-1α of glioma spheres cultured under normoxic and hypoxia microenvironment, we explored the impact of hypoxia on the proliferation of GCSs.(2) By testing the CD133+phenotype, cell vitality of glioma spheres and HIF-1α knockdown glioma spheres under hypoxia microenvironment, we validated the role of HIF-la on the proliferation of GCS cultured under hypoxia microenvironment. Also, by testing the transcription and expression of Notchl signal pathway relative genes, we investigate whether hypoxia, especially HIF-1α modulate Notch signaling to impact the proliferation of GCSs.Methods:(1) Glioma spheres were cultured in normoxic and hypoxia microenvironment for24and48h. Then CD133+phenotype was measured with flow cytometry, cell vitality was measured with CCK8method, mRNA expression of HIF-1α was measured with Real Time-qPCR and protein expression of HIF-1α was measured with Western Blot.(2) We constructed HIF-1α knockdown glioma spheres by silence RNA, then the two glioma spheres were cultured in hypoxia microenvironment for24and48h. CD133+phenotype was measured with flow cytometry, cell vitality was measured with CCK8method. Glioma spheres were cultured in normoxic and hypoxia microenvironment for24h, HIF-1α knockdown glioma spheres were cultured in hypoxia microenvironment for24h. Then cell cycle was evaluated by flow cytometry. The mRNA and protein expression of Notchl signal pathway relative genes were measured with Real Time-qPCR and Western Blot, respectively.Results:(1) Compared to normoxic group, CD133+phenotype, cell vitality and mRNA expression of HIF-1α were significantly elevated in glioma spheres cultured under hypoxia microenvironment.(2) Compared to hypoxia group, CD133+phenotype and cell vitality were significantly reduced in HIF-1α knockdown glioma spheres.(3) Compared to normoxic group, the percentage of G1cell of hypoxia group was significantly reduced, and the percentage of S and G2cell were significantly elevated. Compared to hypoxia group, the percentage of G1cell of HIF-1α knockdown glioma spheres was significantly reduced, and the percentage of G2cell were significantly elevated.(4) Compared to normoxic group, the mRNA and protein expression of Notchl signal pathway relative genes (Notch-1, jaggad-1, HES-1) were significantly elevated in glioma spheres cultured under hypoxia microenvironment. Compared to hypoxia group, the mRNA and protein expression of Notchl signal pathway relative genes were significantly reduced.Conclusions:(1) Hypoxia could promote the proliferation and cell vitality of GCSs and the protein expression of HIF-1α. The silence of HIF-1α could reduce the proliferation and cell vitality of GCSs. It suggested that HIF-1α may play an important role in this process.(2) Hypoxia could induce GCSs enter S phase, and promote the proliferation of GCSs. The silence of HIF-1α induced GCSs cycling arrest at G2/M phases and reduced the proliferation of GCSs.(3) Hypoxia and HIF-1α modulate Notch signaling to promote the proliferation of GCSs. The silence of HIF-1α inhabit the Notch signaling, therefore, the proliferation of GCSs was inhabited either.更多还原