Exploring The Origin Of CNS Hemangioblastoma And The Key Pathogenic Factors

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.