【作者】 谢嵘；

【导师】 朱剑虹； 毛颖；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 背景中枢神经系统血管母细胞瘤(Central Nervous System Hemangioblastoma,CNS HB)是目前神经外科领域治疗较为困难的疾病之一。常伴有其他部位的囊肿或肿瘤,称为VHL病(von Hippel Lindau Disease),危害更大。迄今为止其组织学起源及发病机制仍不清楚,2000年WHO将其归为来源未定肿瘤,而2007年WHO最新的中枢神经系统肿瘤分类中将其归类于脑膜肿瘤中的其他脑膜相关性肿瘤项目下,具体来源亦不明确。因此,我们对CNS HB进行细胞培养、组织病理学观察、分类功能基因芯片研究及蛋白质组学分析。目的建立CNS HB细胞体外培养的方法,在组织、细胞中筛选CNS HB致病关键因素,为明确其组织学起源、阐明发病机制提供依据。方法1.收集复旦大学附属华山医院神经外科2004年9月至2007年6月手术切除并经术后病理证实的CNS HB组织标本35例及正常脑组织15例,均经得患者本人或代理人的知情同意。2.第一部分研究中对其中13例CNS HB应用Ⅱ型胶原酶联合胰蛋白酶消化法进行原代及传代培养,并通过形态学观察、免疫荧光染色、细胞超微结构观察、培养液上清EPO及VEGF浓度测定对培养细胞进行鉴定。3.第二部分研究中对所有收集的CNS HB及正常脑组织标本进行病理切片、常规H.E.染色及选择性的免疫组化染色,比较HB与对照组各个组成细胞之间对每个抗体的染色阳性率及染色强度的差异以及VHL与NVHL(None-VHL Disease)组之间的染色差异。4.第三部分研究中根据前两部分研究结果,对6例VHL CNS HB、7例NVHL CNSHB及7例正常脑组织混合而成的3份样品以及VHL CNS HB、NVHL NS HB及正常神经细胞(共3株细胞)进行RNA抽提,与SuperArray公司(USA)Oligo GEArray(?)干细胞及缺氧信号通路功能基因芯片分别杂交,比较CNS HB组织与正常脑组织、CNSHB细胞与正常神经细胞、VHL与NVHL组之间的差异表达基因。应用Real-time PCR及RT-PCR对结果进行验证。5.第四部分(一)研究中应用2-DE联合MALDI-TOF-MS的蛋白质组学技术对5例CNS HB组织标本及4例正常脑组织标本进行蛋白质提取、双向凝胶电泳(2-DE)、快速银染、差异蛋白质点的选取和质控措施、切胶及蛋白质酶解、MALDI-TOF-MS质谱鉴定,获得与CNS HB致病相关的差异表达蛋白质,并应用免疫组化、RT-PCR及Western Blot自多个层次验证研究结果。6.第四部分(二)研究中应用2D-HPLC串联LTQ-Orbitrap MS的蛋白质组学技术对CNS HB细胞及正常神经细胞进行蛋白质提取、2D-HPLC色谱分离、LTQ-Orbitrap MS质谱鉴定、Shoutgun法定量,比较并筛选与CNS HB致病相关的差异表达蛋白质,并与CNS HB组织蛋白质组学研究结果比较。结果1.第一部分细胞培养研究中,13例CNS HB组织有11例培养成功。形态学观察细胞呈扁平状短梭形或不规则三角形,类似人骨髓基质细胞(MSCs)以集落方式生长。细胞免疫荧光染色显示CD133+、Nestin+、Vimentin+、VEGF+、EPO+、CD34-、SMA-、GFAP-,提示CNS HB具有干细胞的起源。培养液上清检测发现细胞具有向外分泌VEGF及EPO的能力。2.第二部分组织病理学研究中发现Vimentin、CD117、CD133、Nestin、EPO及EPO-R在CNS HB组织的基质细胞、血管内皮细胞、周细胞中均染色阳性,CD34、FVIIIRA及SMA在CNS HB组织的血管内皮细胞、周细胞中染色阳性,在基质细胞中染色阴性。GFAP在CNS HB三种组成细胞中均染色阴性。VEGF仅在基质细胞中染色阳性,而其受体Flt-1仅在血管内皮细胞中染色阳性。VHL则在CNS HB三种细胞中均染色阳性。VHL与NVHL组比较,EPO及EPO-R的染色强度(强阳性率)在VHL组明显高于NVHL组,差异有高度统计学意义。VHL染色阳性率及染色强度在VHL组均小于NVHL组,但差异无统计学意义。其余抗体的染色阳性率及染色强度在VHL组与NVHL组类似,均无统计学差异。Real-time PCR及RT-PCR验证结果与本研究一致。3.第三部分CNS HB组织与细胞的分类功能基因芯片研究中,人干细胞基因芯片的组织与细胞实验中分别发现8个及2个差异表达基因,其中具有相同趋势的差异表达基因有2个:上调1个ABCG2;下调1个RBPSUHL。VHL组与NVHL组之间均无表达差异。人缺氧信号通路基因芯片的组织与细胞实验中分别发现14个及11个差异表达基因,其中具有相同趋势的差异表达基因有6个:上调4个EPO、VEGF、HIF1α、CAⅨ;下调2个HIF1αI、KHSRP。EPO在VHL组较NVHL组明显上调。Real-time PCR及RT-PCR验证可以重复本研究结果。4.第四部分(一)中,首次应用2-DE联合MALDI-TOF-MS蛋白质组学技术建立了HB组与对照组9例组织样品的分辨率及重复性较好的非混合标本2-DE图谱,经ImageMaster 2D软件分析及质控措施后,确定有效差异蛋白质点共12个,HB组较对照组上调7个点,下调5个点。其中仅在HB组表达3个点,仅在对照组表达3个点,HB组较对照组表达增高4个点,下调2个点。12个有效差异蛋白质点在所有9张图谱上经质谱分析、检索MASCOT数据库及生物信息学分析后,全部鉴定成功。其中鉴定所得差异蛋白质Vimentin与CNS HB的干细胞起源有关;CAⅨ与缺氧信号通路有关;14-3-3可能通过抑制细胞凋亡而激活CNS HB细胞;DAAH的缺失加重了血管内皮细胞的功能障碍,最终导致异常血管网的形成,并可能参与CNS HB的囊变过程。而其他蛋白质亦与CNS HB发病密切相关,以往均未见报道。对鉴定的蛋白质点挑选Vimentin、14-3-3、CAⅨ进行免疫组化染色、RT-PCR及Western Blot验证,与本研究结果完全一致。5.在第四部分(二)中,应用2D-HPLC串联LTQ-Orbitrap MS蛋白质组学技术在CNS HB细胞及正常神经细胞株中分别分离了675及532个蛋白质,并全部鉴定成功。这些蛋白质的功能涉及生物学的多个方面。经过Shotgun法定量后发现差异表达蛋白质628个,占蛋白质总数的70.1%。其中仅在CNS HB细胞中表达358个,仅在正常神经细胞中表达215个,CNS HB细胞较正常神经细胞上调30个,下调25个。结果包括在组织实验中筛选获得的11个与CNS HB发病密切相关的差异蛋白质(Hemoglobin beta chain除外)。另外尚发现其他与CNS HB致病相关的蛋白质如PDGFA associated protein 1与干细胞分化有关;Annexin可能是一个新的临床诊断标志物,值得进一步深入研究。CNS HB细胞与正常神经细胞差异较大,可能具有不同的细胞起源。结论1.建立CNS HB细胞体外培养的方法。发现所培养的细胞是CNS HB基质细胞,具有中胚层间质组织干细胞的起源,可能是一种良性肿瘤的干细胞,在体内特定条件下向血管细胞分化并形成肿瘤实质,最终导致CNS HB的发生。2.揭示缺氧信号通路在CNS HB发病中的重要作用。HIF1α是缺氧信号通路最关键的环节。其在CNS HB中的上调导致其下游基因如EPO、VEGF、CAⅨ、KHSRP等表达异常,最终形成CNS HB。EPO及VEGF通过基质细胞的自分泌与旁分泌机制,促使CNS HB发生。3.首次发现EPO是VHL CNS HB与NVHL CNS HB组织细胞中最主要的差异表达因素,提示EPO在VHL病发病过程中起到更大作用,增加了患者罹患肿瘤的几率。4.应用2-DE联合MALDI-TOF-MS蛋白质组学技术对CNS HB肿瘤组织及正常脑组织进行分析,首次建立具有高分辨率及可重复性CNS HB的2-DE图谱,有效筛选并鉴定12个与CNS HB发病密切相关的蛋白质,其中Vimentin与CNS HB干细胞起源有关;CAⅨ则是缺氧信号通路的重要分子,通过直接损伤及局部缺氧导致CNS HB细胞异常分化;14-3-3可能通过抑制细胞凋亡而激活CNS HB细胞;DAAH的缺失加重了血管内皮细胞的功能障碍,最终导致异常血管网的形成。以往均未见报道。5.应用2D-HPLC串联LTQ-Orbitrap蛋白质组学技术对CNS HB细胞与正常神经细胞进行分析,分别分离675及572个蛋白质,且全部鉴定成功。这些蛋白质的功能涉及生物学的多个方面。包括在组织实验中筛选获得的11个与CNS HB发病密切相关的差异蛋白质(Hemoglobin beta chain除外)。在其他发现的与CNS HB发病相关的蛋白质中,PDGFA associated protein 1与干细胞分化有关,Annexin可能是一个新的临床诊断标志物,值得进一步研究。6.CNS HB的发病是多因素、多步骤参与的复杂过程,本研究发现了多个与其致病相关的基因或蛋白质,为深入研究其组织学起源及发病机制提供充分的依据及恰当的切入点。综合上述结论,本研究发现CNS HB可能具有中胚层间质组织干细胞的起源,EPO、VEGF、HIF1α、Vimentin、14-3-3、CAⅨ、DAAH等是其致病的关键因素。更多还原

【Abstract】 BackgroundAt present Central Nervous System Hemangioblastoma(CNS HB) is one kind of the most difficult diseases to treat in the field of Neurosurgery.Sometimes CNS HB is accompanied with other cysts or tumors called VHL disease which has more harmfulness to people.Up to now the histological origin and pathogenesis of CNS HB still remain unclear. In 2000 it was classified into the tumor with an uncertain origin by WHO.In 2007 it was newly classified into the tumors of meninges—other neoplasms related to the meninges.In this research work we have used the methods of cell cultivation,histopathological observation,classified functional gene arrays and proteomics analysis to study CNS HB.ObjectiveOur objective is to establish the approach of CNS HB cell cultivation and screen the key pathogenic factors of CNS HB from CNS HB tissues and cells,so as to provide the key factors and evidences for illuminating the histological origin and pathogenesis of CNS HB.Methods1.In this research work thirty-five CNS HB and fifteen normal brain tissues resected in operation and confirmed by pathological sections were collected from department of Neurosurgery,Huashan Hospital,Fudan University during 2004.9-2007.6.The research was informed consent by all the patients or their attorneys.2.In Part One of the research thirteen CNS HB tissues were primary and passaging cultured using typeⅡcollagenase combined with trypsin digestion.The cells were identified by morphological observation,immunofluorescence,ultramicrostructural observation,EPO and VEGF concentration measurement of culture fluid supernatant.3.In Part Two of the research all the specimen of CNS HB and normal brain tissues were carried out pathological section observation,routine HE stain and selective immunohistochemical stain.The staining positive rate and intensity discrepancy of every single antibody were compared between the composing cells of HB and control groups, VHL and NVHL groups. 4.In Part Three of the research,according to the results of Part One and Part Two,six VHL CNS HB,seven VHL CNS HB,seven normal brain tissues which were admixed into three portions of samples and three cell strains of VHL CNS HB,NVHL CNS HB,normal neural cells were collected and carried out RNA extraction,then were hybridizated with Oligo GEArray(?) classified functional gene arrays(SuperArray,USA) of stem cell and hypoxia signal pathway.The differential expression genes were compared between CNS HB and normal brain tissues,CNS HB and normal neural cells,VHL and NVHL groups and were verified by Real-time PCR and RT-PCR.5.In Part Four(Ⅰ) of the research the total proteins from 5 CNS HB and 4 normal brain tissues were analyzed using 2-DE combined with MALDI-TOF-MS proteomics technology. The samples were progressed total proteins extraction,Two-Dimension Electrophoresis (2-DE),quickly silver staining,discrepant protein spots selection and quality control,spot cutting and protein enzymolysis and MALDI-TOF-MS.Then differential expression proteins were obtained and verified by immunohistochemistry,RT-PCR and Western Blot.6.In Part Four(Ⅱ) of the research the total proteins from CNS HB and normal neural cells were analyzed using 2D-HPLC combined with LTQ-Orbitrap MS proteomics technology.The samples were progressed total proteins extraction,Two Dimension-High Performance Liquid Chromatography(2D-HPLC),LTQ-Orbitrap MS,shotgun quantity. Then differential expression proteins were obtained and compared with those from CNS HB tissues experiments.Results1.In Part One of the research,eleven of thirteen CNS HB tissues were cultivated successfully.Morphological observation suggested CNS HB cells were flat,short fusiform shape or irregular triangle shape.The cells grew as a style of colony just like human mesenchymal stem cells(MSCs).The results of immunofluorescence showed CD133+, Nestin+,Vimentin+,VEGF+,EPO+,CD34-,SMA-,GFAP-,which suggested CNS HB cells had an origin of stem cells.EPO and VEGF concentrations measurement showed the cells had the ability of exocrining EPO and VEGF.2.In Part Two of the research,histological study showed Vimentin,CD117,CD133, Nestin,EPO,EPO-R staining in the stromal cells,endothelial cells and perithelial cells of CNS HB were positive.CD34,FVⅢRA,SMA staining in the endothelial cells and perithelial cells were positive and in the stromal cells were negative.GFAP staining in three kinds of CNS HB cells was negative.VEGF staining was positive only in the stromal cells. Its receptor,Fit-1,was positive only in the endothelial cells.VHL staining in three kinds of CNS HB cells was positive.The staining positive rate and intensity discrepancy of EPO and EPO-R in VHL group were higher than that in NVHL group with statistical significance. The staining positive rate and intensity discrepancy of VHL in VHL group were lower than that in NVHL group,but the difference had no statistical significance.Other antibodies had the same staining results between VHL and NVHL group without statistical significance. The results could be repeated by Real-time PCR and RT-PCR.3.In Part Three of the research,we had screened eight and two differential expression genes using human stem cells gene array from CNS HB tissues and cells.Two of the total differential expression genes had the same tendency both in CNS HB tissues and cells: up-regulated one gene,ABCG2;down-regulated one gene,RBPSUHL.No differences were found between VHL and NVHL groups.Fourteen and eleven differential expression genes had been found using human hypoxia signal pathway gene array from CNS HB tissues and cells.Six of the total differential expression genes had the same tendency both in CNS HB tissues and cells:up-regulated four genes,EPO,VEGF,HIF1α,CAⅨ;down-regulated two genes,HIF1αⅠ、KHSRP.EPO was distinctly up-regulated in VHL group compared with NVHL group.The results could be confirmed by Real-time PCR and RT-PCR.4.In Part Four(Ⅰ),the 2-DE gels of nine samples from HB and control groups with high resolution and reproducibility had been set up using 2-DE combined with MALDI-TOF-MS proteomics technology.After software analysis and quality control,twelve effective discrepant protein spots were selected.Of the total twelve proteins,seven protein spots were up-regulated and five proteins spots were down-regulated in HB group compared with the control group.Total effective discrepant protein spots were carried out MALDI-TOF MS, MASCOT database searching and bioinformatical analysis from all 2-DE gels.And total twelve effective discrepant protein spots were identified successfully.Among them, Vimentin was related to the stem cell origin of CNS liB.CA IX was an important element of hypoxia signal pathway.14-3-3 could induce the CNS HB cell by inhibiting the process of cell apoptosis.DAAH enhanced the functional dysfunction of vascular endothelial cells, led to the abnormal vascular net and participated the procedure of cyst formation.Other proteins were also related to the occurring of CNS HB.There were no reports about these before.The results could be verified by immunohistochemical stain,RT-PCR and Western Blot.5.In Part Four(Ⅱ),675 and 532 proteins were separated and identified from CNS HB and normal neural cells using 2D-HPLC combined with LTQ-Orbitrap MS proteomics technology.The function of identified proteins involved in lots of aspects of biology.After shotgun quantity,628 differential expression proteins(70.1%of total proteins) were found. Among them 358 proteins were exclusively found in CNS HB cells and 215 proteins found in normal neural cells.30 proteins were up-regulated and 25 proteins were down-regulated in CNS HB cells compared with normal neural cells.The proteins included eleven proteins simultaneously discovered from the experiments of CNS HB tissues and other newly found proteins.Of the total newly found proteins,PDFGA associated protein 1 was related with the stem cell differentiation.Annexin might be a new clinical diagnostic marker,which was worth of further study.The proteins of CNS HB cells had distinguished differences from those of normal neural cells which suggested CNS HB and normal neural cells had a distinct histological origin.Conclusions1.In this research work we have established the approach of CNS HB cell culture in vitro and found the stromal cell of CNS HB is the tumor cell indeed.The stromal cell of CNS HB has an origin of mesodermal mesenchymal stem cell and may be a kind of stem cell of benign tumors.It can differentiate towards the endothelial cell and perithelial cell in vivo which lead to the occurring of CNS HB.2.The hypoxia signal pathway plays an important role in CNS HB and the up-regulation of HIF1αleads to the abnormal expression of EPO,VEGF,CAⅨ,KHSRP,et al.The autocrine and paracrine mechanism of EPO and VEGF facilitates the formation of CNS HB.3.EPO is the most primary differential expression factor between VHL CNS HB and NVHL CNS HB and has a much more influence on VHL disease occurring.EPO increases the morbidity of CNS HB and VHL disease.4.In this research work we have firstly successfully set up the 2-DE gels from CNS HB tissues and cells with high resolution and reproducibility and effectively screened the proteins related with CNS HB occurring using 2-DE combined with MALDI-TOF-MS proteomics technology,including Vimentin,CAⅨ,14-3-3,DAAH,et al,which provide the sufficient evidences for the histological origin and pathogenesis of CNS HB.Among them,Vimentin was related to the stem cell origin of CNS HB.CAⅨwas an important element of hypoxia signal pathway.14-3-3 could induce the CNS HB cell by inhibiting the process of cell apoptosis.DAAH enhanced the functional dysfunction of vascular endothelial cells,led to the abnormal vascular net and participated the procedure of cyst formation. 5.We have firstly analyzed the proteins from CNS HB and normal neural cells using 2D-HPLC combined with LTQ-Orbitrap MS proteomics technology.675 and 532 proteins were successfully separated and identified.The function of identified proteins involved in lots of aspects of biology.The proteins included eleven proteins simultaneously discovered from the experiments of CNS HB tissues and other newly tbund proteins.Of the total newly found proteins,PDFGA associated protein 1 was related with the stem cell differentiation. Annexin might be a new clinical diagnostic marker,which was worth of further study.6.CNS HB occurring is an intricate,multicomponent,multifactorial and multi-step process which is mediated by a variety of proteins.In this research work,partial genes and proteins related with CNS HB have been found which provide sufficient evidences and correct investigation directions for the histological origin and pathogenesis of CNS HB.Synthetizing the conclusions,this study suggests that CNS HB has an origin of mesodermal mesenchymal stem cell.EPO,VEGF,HIF1α,Vimentin,14-3-3,CAⅨ,DAAH, etc,are the key pathogenic factors of CNS HB.更多还原