【作者】 吴银艳；

【导师】 黄强；

【作者基本信息】 苏州大学 ， 神经外科学， 2008， 博士

【摘要】 第一部分胶质瘤干/祖细胞长期培养及其特征分析目的:以短期培养为主的人胶质瘤干/祖细胞(human Glioma Stem Cells/ Progenitors,hGSCPs)研究进展迅速,但各家报告的生物学特征不尽一致,建立一个生物学特征相对稳定的永生化细胞系势在必行,可为深入探讨胶质瘤干/祖细胞生物学特征奠定基础。方法:将同一患者初发胶质瘤和复发的播散灶内的肿瘤组织制成单细胞,用CD133免疫磁珠分选阳性细胞,在干细胞培养液中进行长期传代培养和实时液氮冻存、复苏后再培养。并根据无限增殖、多向分化等干细胞必备的指标进行鉴定。再用细胞扩增、分化走向、分子和细胞遗传、致瘤性等指标观察其生物学特征。结果:从原发和复发的胶质瘤组织中分离得到的CD133+细胞,分别在体外连续培养了58个月和64个月,液氮冻存后复苏仍能保持无限增殖和多向分化的特征不变。在无血清干细胞培养基中呈球状生长,能扩增、少分化;在含血清培养基中培养能分化成多形性瘤细胞,贴壁生长无接触抑制。免疫共聚焦结果显示:肿瘤球体中CD133+细胞占少数,Nestin+细胞占多数;贴壁分化细胞中Nestin+细胞占绝对多数,即使有GFAP+和β-TubulinIII+细胞出现,但都共表达Nestin。球体细胞、贴壁细胞的DNA和染色体显带都为异倍体。肿瘤球裸小鼠原位移植瘤原发为局部侵袭性生长,而复发者为播散性生长,并广泛浸润软脑膜。结论:建立了来自同一患者两株能在体外长期传代培养,保持无限增殖、多向分化潜能不变的人胶质瘤干/祖细胞系,不仅有利于进一步研究胶质瘤发生、发展、侵袭的特性和机制,还为研究其播散性复发等机制奠定了基础。第二部分原发和复发胶质瘤干/祖细胞在裸小鼠脑内移植瘤的特征目的:人源胶质瘤干/祖细胞(human Glioma Stem Cells/ Progenitors,hGSCPs)能分化成亲本肿瘤细胞的全部亚型,在免疫缺陷动物脑内具有高致瘤性已得到公认,但其侵袭性与亲本肿瘤的关系以及GSCPs与移植瘤组织中血管细胞的关系尚未明确。本研究探讨源自同一患者原发和复发肿瘤组织的GSCPs在裸小鼠颅内原位移植瘤的特征以及GSCPs移植瘤组织中的肿瘤细胞和血管内皮细胞的关系,旨在证明GSCPs裸小鼠原位移植瘤的侵袭性与亲本肿瘤的关系和GSCPs在脑肿瘤组织重构中所起的作用。方法:将已在体外连续传代培养的原发和复发hGSCPs球在立体定向仪辅助下接种到裸小鼠右脑尾状核。实验鼠出现恶病质时处死,取移植瘤组织常规石蜡包埋、切片、HE、人类白细胞抗原(human leucocyte antigen,HLA)和基质金属蛋白酶-1(matrix metalloproteinase-1,MMP1)染色,观察其致瘤率、侵袭性和血管来源细胞。结果:所接种的40只裸小鼠在30天内全部因肿瘤而发病。无论是原发还是复发hGSCPS在裸小鼠脑内接种部位呈浸润性生长的同时,还散布于其它区域。但两者的侵袭形式不尽相同,原发者在接种侧半球,呈团块,局限于脑实质;复发者,还浸润软脑膜和对侧半球,呈粟粒样分布。MMP1+细胞的分布,复发者比原发者要密集得多。HLA染色显示移植瘤中存在人源肿瘤细胞直接构成的血管。结论:hGSCPs除了具有高致瘤性,还具有高侵袭性。复发的hGSCPs侵袭性更强,除了与MMP1高表达有关,还说明hGSCPs在侵袭性上具有亲本肿瘤特异性。在hGSCPS的裸小鼠移植瘤中,肿瘤细胞在宿主脑内广泛分布并构成了血管。GSCPs可能转分化成肿瘤血管细胞,在脑胶质瘤组织重构中起重要作用。第三部分克隆基因工程鼠的胶质瘤干/祖细胞和神经干/祖细胞目的:据推测胶质瘤干/祖细胞(Glioma Stem Cells/ Progenitors,GSCPs)源于神经干/祖细胞(Neural Stem Cells/ Progenitors,NSCPs),但缺少实验根据。克隆已经发生胶质瘤的基因工程鼠的GSCPs和NSCPs以及同品系胎鼠的NSCPs,旨在为进一步研究两者渊源关系提供实验用细胞。方法:取Tet-On小鼠位于小脑和大脑交界区域内的肿瘤组织和位于远离肿瘤的侧脑室室管膜下及其周围的正常脑组织,分别制成单细胞悬液,分别培养于无血清干细胞培养基和含血清培养基中。相差显微镜观察细胞形态,电镜观察其超微结构,流式细胞仪检测Hoechst33342阴性的SP细胞、细胞周期和染色体倍体,免疫荧光检测球体及其分化细胞中的标志物表达情况。结果:源于Tet-On鼠的肿瘤组织和室管膜区正常组织以及胎鼠脑组织细胞,在无血清培养基条件下,呈悬浮的球体状生长,并且表达Nstin;在含血清的培养条件下,贴壁分化成能表达胶质细胞和神经元标志蛋白的细胞。细胞球体在体外已传代。电镜结果显示,GSCPs球体细胞具有核质比高、细胞器不发达的幼稚细胞特征。GSCPs球体中SP细胞比例为17.27%,NSCPs球体中SP细胞比例为2.48%,GSCPs球体中异倍体细胞比例为26.77%,NSCPs球体中异倍体细胞比例为0%。结论:分别在Tet-On胶质瘤模型鼠中克隆到的GSCPs和室管膜区域的NSCPs,都能在体外连续传代培养,为进一步研究两者的细胞分子生物学关系奠定了基础。第四部分胶质瘤干/祖细胞的分子信息分析目的:已知胶质瘤干/祖细胞(Glioma Stem Cells/ Progenitors,GSCPs)与神经干/祖细胞(Neural Stem Cells/ Progenitors,NSCPs)在分子信号传递通路开通和关闭时程调控上存在差异,而这种差异有可能就是NSCPs突变成GSCPs的分子原因。通过基因表达谱分析,筛选其差异分子,为进一步研究胶质瘤发生发展的分子病因奠定基础。方法:取Tet-On胶质瘤模型鼠的肿瘤组织中克隆到的GSCPs和室管膜区域的NSCPs以及来自于胎鼠胎脑的NSCPs,常规抽提和纯化RNA,用Affymetrix Mouse Expression Array 430 2.0基因表达谱芯片检测三种细胞的基因表达谱改变,进行两两对比筛选出上调2倍及以上的基因和下调2倍及以上的基因得到三组数据。将初筛得到的三组差异表达基因分别进行cell-cycle、Wnt、Notch和TGF-βpathway的分析。结果:通过对三种细胞的基因表达谱进行两两比较,经初步筛选后上调2倍及以上的基因分别有3423条、1014条和2441条;下调2倍及以上的基因分别有2524条、2546条、4266条。cell-cycle、Wnt、Notch和TGF-βpathway分析结果显示cell-cycle pathway在三组数据中都有较多上调和下调的基因,而Notch pathway中的基因变化主要是下调,且变化的基因较少。结论:生物信息学是分析基因表达谱的有力工具,本实验对比了三种细胞基因表达的差异,分析了cell-cycle、Wnt、Notch和TGF-βpathway中的变化基因,为以后的验证工作和深入研究提供了线索。更多还原

【Abstract】 Part I Long-term Culture and Characteristics of Glioma Stem Cells/ProgenitorsObjective: Most current research on human brain tumors is focused on the short term cultured human Glioma Stem Cells/ Progenitors (hGSCPs). The biological characteristics of these hGSCPs in most reports were different. Therefore, Establishment of a permanent GSCPs line will provide a powerful tool to investigate the biological characteristics of GSCPs.Methods: GSCPs derived from the primary and recrudescent brain tumor tissues of the same patient respectively. CD133-positive cells were isolated by the Miltenyi Biotec CD133 cell isolation kit .The cells were culured in medium of neural stem cell, passaged , frozen in liquid nitrogen, resuscitate and re-cultued. The capability of indefinite proliferation and multi-directional differentiation were identified. The cell amplification, cell differentiation, molecular genetics and cytogenetics were investigated.Results: CD133-positive cells derived from the primary and recrudescent human brain tumor tissue were maintained for 58 months and 64 months. In serm-free medium, the cells can form tumor spheres and possess the feature of long-term proliferation and seldom differentiation; in medium contained serm they attached to the culture dishes and can differentiate polymorphic tumour cell. In tumor spheres, minority are CD133-positive and majority are Nestin-positive. In adherent differentiated cells, majority are Nestin-positive and some coexpress GFAP orβ-TubulinIII and Nestin. The chromatosomes of cell speres and adherent cells both are heteroploid. Nude mice intracranial xenograft tumors of tumor spheres derived from the primary brain tumor tissues showed invasive growth in injected location and derived from recrudescent brain tumor tissues were disseminated foci and generally infiltrated meningina.Conclusion: Establishment of two GSCPs lines that are able to maintain the properties of indefinite proliferation, multi-directional differentiation and disseminated will lay the foundation to deeply investigate the mechanisms of glioma tumorigenesis ,growth, invasion , disseminateion .Part II Characteristics of Primary and Recurrent Human Glioma Stem Cells/Progenitors Transplantation Tumors in Nude Mice IntracalvariumObjective: Human Glioma Stem Cells/ Progenitors (hGSCPs) can differentiate into all subtypes of parent tumor cells and have high tumorigenicity in nude mice, but the relationship transplantation tumor invasiveness and parent tumor and the relationship of GSCPs and vascular endothelial cells in tumor tissues is still unknown. To elucidate the high invasiveness of hGSCPs and the role of hGSCPs in brain tumor tissue remodeling, we investigated characteristics of primary and recurrent hGSCPs transplantation tumors in nude mice intracalvarium and the origin of vascular endothelial cells in GSCPs intracranial xenografts.Methods: Primary and recurrent hGSCPS spheres were injected into the right caudate nucleus of nude mice. The mice were killed at the time of cachexia. Xenograft tissues were obtained, performed routine paraffin embedding, sliced and conducted HE staining, human leucocyte antigen(HLA)staining and matrix metalloproteinase-1(MMP1) staining. The tumorigenesis ratio, invision and the origin of vascular endothelial cells were observed under light microscope. Results: All forty injected nude mice formed tumor in 30 days. Xenografts show invasive growth in injected location and spread other area. The invision of xenografts of primary and recurrent GSCPs is different. Primary GSCPs xenografts were concentrated in injected hemicerebrum. Recurrent GSCPs xenografts displayed dissemination growth, even to reach hemicerebrum and cerebral pia mater of opposite side. Minority of primary GSCPs xenograft cells are MMP1-positive and majority of recurrent GSCPs xenograft cells are MMP1-positive.In HLA staining, tumor cell-derived vascellums were observed.Conclusion: HGSCPs have strong oncogenicity and invasiveness. The more invasiveness of recurrent hGSCPs could relate to high expression of MMP1. In the intracranial xenografts of GSCPs, tumor cells widely distributed and constructed vascellums. The GSCPs could play an important role in glioma tissue remodeling by differentiating into tumor vascular endothelial cells.Part III Isolation and Culture of Glioma Stem Cells/ Progenitors and Neural Stem Cells/Progenitors in Genetically Engineered Mouse Brain TumorObjective: Glioma Stem Cells/Progenitors(GSCPs) could derive from Neural Stem Cells/ Progenitors (NSCPs). To deeply investigate the relationship of GSCPs and NSCPs, we isolated and cultured GSCPs and NSCPs from genetically engineered mouse and NSCPs from embryo mouse.Methods: Cells from brain tumor tissue and normal brain tissue under ependyma of lateral ventricle away from tumor of genetically engineered mouse and brain tissue of embryo mouse were cultured in serum-free medium and serum medium, respectively. The cells were obeserved under phase-contrast microscope. The ultrastructures of GSCPs spheres were obeserved under transmission electron microscope (TEM). Hoechst 33342 was used to sort for the side population (SP) phenotype. Cell cycle and chromosome ploidy were performed on Flow cytometry. Multiple immunofluorescence was used to assays the cell markers of GSCPs spheres and differentiated cells.Results: All cells from genetically engineered mouse brain tumor tissue and nomal brain tissue and embryo mouse brain tissue formed stem-like spheres when cultured in serum-free medium and express Nestin, and differentiated into neuronal and glial cells when cultured in medium containing 10% FCS. The spheres were passaged in vitro.The ultrastructures of GSCPs sphere indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. The average first sort percentage of SP cells was 17.27% in GSCPs spheres and 2.48% in NSCPs spheres. 26.77% cells in GSCPs spheres cells and 0% cells in NSCPs spheres were heteroploid.Conclusion: Isolatin and culture of cells from brain tumor tissue and normal brain tissue under ependyma of lateral ventricle away from tumor of genetically engineered mouse and brain tissue of embryo mouse will lay the foundation to deeply investigate molecular biology relationship of GSCPs and NSCPs.Part IV Molecular information assays of Glioma Stem Cells/ ProgenitorsObjective: The difference in molecular signal pathway of Glioma Stem Cells/ Progenitors (GSCPs) and Neural Stem Cells/ Progenitors (NSCPs )could be the reason the transformation from NSCPs into GSCPs. The differential genes of gene expression profile will lay the foundation to deeply investigate molecular etiopathogenisis of gliomas formation and development.Methods: Spheres of GSCPs and NSCPs from genetically engineered mouse brain tumor tissue and nomal brain tissue and embryo mouse brain tissue were collected. RNA was extracted and purified. The Affymetrix Mouse Expression Array 430 2.0 gene chips were used to detected the significant gene expression changes. After selected the genes of up-regulation and down regulation greater than or equal to 2 folds, cell-cycle, Wnt, Notch and TGF-βpathway analysis were perform.Results: Up-regulation of genes greater than or equal to 2 folds were 3423 genes,1014 genes and 2441 genes, down regulation genes greater than or equal to 2 folds were 2524 genes,2546 gene and 4266 genes, respectively. Cell-cycle, Wnt, Notch and TGF-βpathway analysis display the changed genes in cell-cycle pathway were more than others and the changed genes and in Notch pathway were less than others.Conclusion: Bioinformatics is powerful tool to analyze gene expression profile. Cell-cycle, Wnt, Notch and TGF-βpathway analysis of three gene chips will provide powful tool to deep investigation.更多还原