【作者】 刘伟；

【导师】 庞琦；

【作者基本信息】 山东大学 ， 神经外科， 2011， 博士

【摘要】 研究背景和目的胶质瘤是成人颅脑肿瘤中最常见的一种恶性肿瘤,发生率约占颅内肿瘤的35.12-61.10%。其生长迅速,具有很强的侵袭性,目前为止,各种手术方法都无法做到肿瘤的完全切除。另外,由于血脑屏障的存在,使得目前诸多的治疗方法诸如化疗或者放疗等都难以取得令人满意的治疗效果。即使进行系统的治疗,患者也往往在术后1年内复发。因此,胶质瘤患者的预后非常差,中位生存期只有12-18个月,长期存活的病例非常罕见。由于大脑淋巴系统非常不发达以及完整的血脑屏障对炎症细胞的阻碍作用等,大脑一直以来被认为是免疫豁免器官。但是最近的研究表明,在健康人和炎症状态下,大脑的免疫监督是存在的,在胶质瘤细胞降低到一定程度时,人体自身的免疫系统可以杀死残存胶质瘤细胞,从而减少胶质瘤的复发。虽然巨噬细胞和小胶质细胞等免疫细胞具有很强的肿瘤渗透性,但是其在胶质瘤中的功能往往是被抑制的。这种抑制作用一般认为是由于巨噬细胞和小胶质细胞中的STAT3信号通路被胶质瘤通过某种途径激活,从而抑制了免疫细胞的功能发挥。但是,胶质瘤是如何激活免疫细胞中的STAT3信号通路目前为止还不是很清楚。因此,寻找可以激活胶质瘤中免疫系统的活性因子,使人体自身的免疫系统发挥正常的防卫功能,便成为治疗肿瘤,改善肿瘤预后的一种重要方法和途径。C-Met属于跨膜受体蛋白,是一种重要的肿瘤标记物,定位于7号染色体长臂2区1带至3区1带,横跨120Kb。C-Met通过与其配体肝细胞生长因子(HGF)结合,对肿瘤的发生,转移和预后起着关键的作用。C-Met在胶质瘤,肝癌,结肠癌等多种肿瘤中呈高表达并且与肿瘤的预后密切相关。S1OOB是一种Ca2+结合蛋白,在星形细胞,成熟少突细胞以及多种肿瘤细胞中均有不同程度的表达。人类S100B基因定位于21号染色体长臂2区2带上。S100B通过与其受体RAGE结合,从而影响神经系统中的神经元细胞,星形细胞以及小胶质细胞。RAGE信号通路的激活可以激活STAT3信号通路。而作为RAGE配体的S100B在胶质瘤中呈现高表达。已有研究表明,S100B在肿瘤的发生和发展中可以抑制机体免疫细胞的激活,从而降低机体的免疫反应,促进肿瘤的生长和复发。但是,S100B具体通过那些途径抑制免疫细胞的激活,以及S100B表达量与免疫抑制程度的关系,目前为止还不是十分清楚。本论文分为两部分：第一部分：通过对C-Met与胶质瘤预后相关性的研究,明确C-Met在胶质瘤预后中所起的重要作用,为以其为靶点改善胶质瘤预后提供依据。第二部分：通过对S100B在RAGE和STAT3信号通路中的功能研究,明确S100B在两个信号通路中所起的作用,初步阐明S100B通过何种途径抑制小胶质细胞等免疫细胞激活的机制,探讨以S100B为靶点,激活颅内的免疫系统,从而抑制肿瘤生长,减少肿瘤复发的胶质瘤治疗新途径。第一部分c-Met在胶质母细胞瘤患者中的表达及其与胶质瘤预后的相关性研究目的：通过检测初发和复发胶质母细胞瘤患者肿瘤标本中c-Met的表达差异,探讨c-Met与胶质瘤预后的相关性。方法：选取19例山东大学附属省立医院神经外科收治的复发胶质母细胞瘤患者为研究对象。收集患者两次手术中胶质瘤组织标本,利用免疫组织化学的方法而来检测c-Met在胶质瘤中的表达。分析c-Met表达量的差异与胶质母细胞瘤无症状生存时间(手术后复发时间)的相关性。结果：1.免疫组织化学结果显示：c-Met的表达量在复发胶质母细胞瘤中明显高于初发患者。2.在初发患者里,肿瘤标本检测c-Met呈高表达则手术后复发时间明显短于c-Met低表达患者。结论：复发胶质母细胞瘤患者中c-Met的表达明显增大,而且c-Met的表达量与胶质瘤患者术后肿瘤复发时间密切相关。因此,以c-Met为靶点在胶质瘤的诊断和预后判断上具有重要的临床意义。第二部分S100B在胶质瘤中对颅内免疫功能的抑制作用及其在STAT3信号通路中可能的作用机制研究目的：先前的研究表明,RAGE信号通路的激活可以激活STAT3信号通路。作为RAGE配体的S100B在胶质瘤中呈现高表达。因此,我们假设：胶质瘤中巨噬细胞和小胶质细胞中STAT3信号通路的激活是由于S100B与其受体RAGE相互作用的结果。通过对S100B在RAGE和STAT3信号通路的研究,明确S100B在两个信号通路中所起的作用,初步阐明S100B通过何种途径抑制小胶质细胞等免疫细胞激活的机制,探讨以S100B为靶点,激活颅内的免疫系统,从而抑制肿瘤生长,减少肿瘤复发的胶质瘤治疗新途径。方法：1.S100B在胶质瘤中的表达以及对RAGE信号通路的影响：1.1用Elisa方法检测S100B在GL261胶质瘤细胞系中的表达；用流式细胞术(FCM)和荧光免疫组织化学的方法检测S100B在胶质瘤动物模型中的表达。1.2用RT-PCR和Western Blotting的方法检测GL261细胞培养液(含有S100B)以及不同浓度的S100B对N9细胞系以及原代骨髓单核细胞中RAGE表达的影响。1.3用流式细胞术和荧光免疫组织化学的方法检测胶质瘤动物模型中RAGE的表达。2.S1OOB对STAT3信号通路的影响：用RT-PCR, Western blotting和FCM的方法检测GL261细胞培养液以及不同浓度的S100B对N9细胞系以及原代骨髓单核细胞中STAT3表达的影响。3. RAGE信号通路与STAT3信号通路的相互联系：用RAGE抗体在体内和体外分别阻断RAGE信号通路,再分别用EMSA, RT-PCR的方法检测STAT3信号通路的变化。结果：1.GL261条件培养基(GCM)可以调节小胶质细胞分泌RAGE我们发现：GCM可以在几个小时内上调RAGE的表达,培养一段时间后(24-48h), RAGE水平是下降的。与此同时,细胞培养上清液中分泌性的RAGE蛋白却是增加的。这种现象可能是由于N9细胞膜上全长RAGE蛋白的断裂所致。最后,我们发现加入GCM共培养48h后RAGE mRNA的表达是上调的。这可能是对蛋白分泌的补充性上调。2. RAGE蛋白在颅内胶质瘤的表达流式细胞术结果显示：在颅内胶质瘤中,超过70%的表达EGFP细胞为MPs(CD11b+, CD45high)和MG(CD11b+, CD451ow)。在载瘤小鼠脑组织和血液中RAGE+CD11b+(MG)细胞出现的频率明显高于正常对照组老鼠。免疫组织化学结果显示：在正常小鼠中,RAGE的表达很低,但是,在载瘤小鼠的肿瘤边界周围RAGE的表达明显增多。3.胶质瘤中S100B的表达将N9细胞与GCM共培养48h内,S100B的分泌量明显增加。在体内试验中,我们发现在胶质瘤细胞内和细胞外间隙中(ECS)都有S100B的表达。而且,大量的MG/MPs细胞也都有S100B的表达。4.低浓度的S100B可以刺激RAGE的表达与GCM的作用类似,S100B在低浓度可以上调RAGE蛋白和mRNA的表达。但是,高浓度的S100B也可以增加N9细胞中分泌性RAGE蛋白的表达。5.GCM和S100B抑制MAPK的激活和MG细胞因子的表达GCM和低浓度的S100B不仅可以抑制N9细胞里rNF-α和IL-1β的基础分泌,还可以抑制经过LPS刺激后的IL-1β分泌。类似的现象也在BMM细胞中得到证实。6.低浓度的S100B可以诱导小胶质细胞中STAT3的表达与GCM相似,低浓度的S100B可以上调STAT3和磷酸化的STAT3的表达。虽然高浓度的S100B也增加了STAT3的转录,但是却没有增加其蛋白表达。并且对磷酸化的STAT3的激活作用也相对温和。这提示我们：胶质瘤内环境中低浓度的S100B可以通过激活STAT3信号通路来调节MG/MP的炎症反应。7.阻断RAGE通路可以抑制STAT3的活性阻断RAGE通路后可以部分抑制STAT3的表达和抗炎因子IL-10的表达。作为对比的是,用对照IgG阻断RAGE从而抑制STAT3的结果没有aRAGE明显。这暗示胶质瘤介导的MG/MP中STAT3的激活可能部分是由于对RAGE信号通路的调节引起的。与体外实验结果相一致,体内试验也证实了aRAGE对pSTAT3的部分抑制作用。结论：体内、体外实验证实：胶质瘤中MG/MP细胞中STAT3信号通路的激活很可能是由于RAGE通路激活引起的。胶质瘤可以分泌低浓度的S100B,作为RAGE配体的S100B可以激活STAT3信号通路从而抑制小胶质细胞的活性。因此,胶质瘤中S100B-RAGE的相互作用在STAT3信号通路的激活和MG/MP细胞活性抑制中起着十分关键的作用。以S100B为靶点的新的免疫系统激活疗法有可能成为肿瘤治疗和改善预后的新途径。更多还原

【Abstract】 Background and ObjectiveThe incidence of malignant gliomas, which is the most common type of primary brain neoplasm, is about 35.12-61.10%. Rapid tumor growth, invasive nature, and presence of the blood-brain barrier, which limits the penetration of large molecules into the central nervous system (CNS), all contribute to a poor response to conventional therapies. Patients with malignant gliomas always get recurred in 1 year despite comprehensive treatments were given to them. Postoperative radiation therapy and chemotherapy prolong the survival time; however, it yields a median survival time of less than 1 year. And long-time survival is extremely rare. Furthermore, brain has been considered to be an immune-privileged site, in part due to lack of lymphatic system and poor penetration of inflammatory cells across an intact blood-brain barrier. Although, recent work has demonstrated the presence CNS immune surveillance in healthy and inflammatory conditions, glioma microenvironment may provide another barrier that attenuates the host anti-tumor response through secretion of inhibitory factors and attraction of immunosuppressive leukocytes. Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is still not clear.Therefore, it is very important to explorer new cytokines which can activate the immune system in gliomas.One of the most interesting tumor markers is the tyrosine kinase receptor c-Met, a transmembrane receptor protein. In humans, the c-Met gene is located on chromosome 7, q21-q31, and spans more than 120 kb in length. c-Met plays a key role in the tumorigenicity, malignant progression and tumor angiogenesis by binding with hepatocyte growth factor (HGF),which is one of its ligands. S100B, a member of a multigenic family of Ca2+ binding protein, has been detected in astrocytes, mature oligodendrocytes and many kinds of tumor cells. In humans, the S100B gene is located on chromosome 21, q22. Once released, S100B can differentially affect neurons, astrocytes, and MG mostly through engagement of RAGE (Receptor for Advanced glycation end products), its primary receptor. The activation of RAGE signaling pathway can activate the STAT3 signaling pathway, which can inhibit the immune system in the brain. However, the exact mechanism by which S100B induce STAT3 activation in MPs and MG is still not clear.This paper was divided into two parts:1. Explore the difference of c-Met expression between primary and recurrent gliomas, and to determine whether the dysregulation of c-Met expression has a role in the malignant progression of gliomas.2. S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas. A better understanding of this interaction may be beneficial in optimizing immunotherapy approaches against malignant brain tumors.The first part:c-Met expression is associated with time to recurrence in patients with glioblastomaObjective:Explore the difference of c-Met expression between primary and recurrent gliomas, and to determine whether the dysregulation of c-Met expression has a role in the malignant progression of gliomas.Methods:19 Chinese patients from the Department of Neurosurgery, Provincial Hospital affiliated to Shandong University between September 2000 and October 2007 were included. Paired primary and recurrent glioma specimens from the same patient were evaluated using immunohistochemical analysis. The association between c-Met expression and progression-free survival time (PFST) was analyzed by using the Kaplan-Meier method and confirmed using the logrank test.Results:1. c-Met overexpression was found in significantly more recurrent glioma (15/19) than primary glioma (7/19; p= 0.020). Recurrent glioma specimens had obviously higher expression levels of c-Met. Age and sex had little effect on the statistical comparisons.2. The seven patients who had primary glioma overexpressing c-Met had a median PFST of 6.1 months (95% CI:3.7-8.6 months), while patients with primary glioma with little or no c-Met expression had a median PFST of 11.5 months (95% CI:6.9-16 months; log-rank test: p=0.026).Conclusion:The results demonstrate that recurrent GBM tend to express c-Met at a higher level than the primary tumors, and that c-Met overexpression is significantly associated with PFST. c-Met may be a useful target in the diagnosis and treatment of gliomas.The second part:S100B Attenuates Microglia Activation in Gliomas:Possible Role of STAT3 PathwayObjective:Low levels of S100B inhibited MG function and induced STAT3 expression. Inhibition RAGE (S100B receptor) with blocking antibody (Ab) abolished STAT3 activation in N9 MG in vitro and MG/MPs in vivo. These findings suggest that S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas. A better understanding of this interaction may be beneficial in optimizing immunotherapy approaches against malignant brain tumors.Methods:1.1 Detect the expression of S100B in GL261 cell line using Elisa; Explore the expresson of S100B in GL261 tumor bearing mice using FACS and immunohistochemical method (IHC).1.2 Explore the changes of RAGE expression in N9 cell line and Bone Marrow Monocyte which were treated with GL261 Condition Medium and different doses of S100B by RT-PCR and WB.1.3 Explore the expression of RAGE in Tumor Bearing mice using FACS and IHC.2. Explore the changes of STAT3 expression in N9 cell line and Bone Marrow Monocyte which were treated with GL261 Condition Medium and different doses of S100B by RT-PCR, WB and FACS.3. Explore the changes of STAT3 signaling pathway after the blocking of RAGE signaling pathway using RAGE antibodies in vitro and vivo. Results:1. S100B Production by GliomasS100B concentration in GCM was 0.22 ng/mL, while in N9 CM was negligible and similar to culture medium (0.02-0.05 ng/mL). Compared with normal brains where very little S100B staining was detected, S100B appeared both intracellularly and in the ECS of gliomas.2. GCM Modulates MG RAGE Expression3. Similar to GCM, S100B upregulated RAGE protein and mRNA expression at very low concentrations.4. S100B upregulated the RAGE expression in tumor bearing mice.5. GCM and S100B Inhibit MAPK Activation and Suppress MG Cytokine Expression6. Low levels of S100B induce STAT3 in MG.7. Blockage of RAGE inhibits STAT3 activity.Conclusion:In summary, we demonstrated that glioma-mediated activation of STAT3 in MG/MP may partly occur through the RAGE pathway. Furthermore, low levels of S100B, a RAGE ligand that is expressed by gliomas, induced STAT3 and inhibited MG activation. These findings suggest that S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas.更多还原