【作者】 李英斌；

【导师】 夏春林；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 目的：通过Tet-on调控系统同时诱导表达c-myc、SV40Tag基因,建立更加稳定的具有干细胞特性的可调控的脑肿瘤小鼠模型,对脑肿瘤干细胞和神经干细胞进行比较研究,旨在为脑肿瘤发病机制的深入研究提供有意义的实验资料。从该模型小鼠诱导前后的室管膜下区及肿瘤组织中,分别分离鉴定正常神经干细胞(neural stem cells)、诱导后神经干细胞(NSC’s)和肿瘤干细胞(brain tumor stem cells, BTSCs)。通过三者的比较研究了解三者的异同点,确定存在差异的分子机制,为脑肿瘤干细胞起源于神经干细胞的假说提供更多依据。方法：将pTRE2-c-myc转基因小鼠与Tet-on、pTRE2-SV40Tag转基因小鼠进行交配,将子代中同时表达pTRE2-c-myc、Tet-on和]pTRE2-SV40Tag的转基因小鼠,进行强力霉素(四环素类)诱导,观察脑肿瘤的发生。通过磁共振成像、大体标本、组织切片、免疫组化对模型鼠脑肿瘤的特征进行研究。从未经强力霉素诱导的Tet-on转基因成年小鼠以及诱导发生脑肿瘤的转基因成体小鼠室管膜下区取材,制成单细胞悬液,培养于无血清干细胞培养基(DMEM/F12,添加bFGF、EGF和B27)以及含血清分化培养基(DMEM+10%FCS)。从出现临床症状的转基因小鼠松果体区肉眼可见的肿瘤组织中取材,制成单细胞悬液,培养于含生长因子的无血清培养基和含血清培养基。用相差显微镜观察正常神经干细胞球体,诱导后神经干细胞球体和肿瘤干细胞球体及其分化细胞形态,细胞增殖情况,电镜观察其超微结构,用流式细胞仪检测三组细胞中Hoechst33342阴性的SP (side population)细胞,细胞周期和染色体倍体。通过免疫荧光,检测球体及其分化细胞中的nestn、NSE、GFAP、MAP2表达情况。通过免疫荧光,比较脑肿瘤干细胞和诱导前后神经干细胞p53, c-myc通路的表达差异。结果：(1)73只同时表达pTRE2-c-myc、Tet-on和pTRE2-SV40Tag的转基因小鼠,经过强力霉素诱导后,30只小鼠发生脑肿瘤,发生率为41.1%。除1例脑干肿瘤外,均位于松果体区,诱导后到发生脑肿瘤的时间最长184d,最短为64d,平均为95d。肿瘤在T1加权像表现为等信号或稍高信号,在T2加权像表现为等信号或稍低信号,增强后可表现为均一强化或不均一强化。病理诊断为未分化成神经管细胞瘤,肿瘤具有高侵袭性。免疫组化显示,肿瘤高表达c-myc、SV40Tag和干/祖细胞标志物nestin。(2)未诱导成体转基因小鼠和诱导发生脑肿瘤的成体小鼠室管膜下区细胞,在无血清培养条件下,均呈悬浮的球状生长。未诱导神经干细胞(NSCs)球体,在含血清培养条件下,贴壁并分化为胶质细胞、神经元。诱导后的神经干细胞(NSC’s)球体,则表现为增殖活性增加和分化抑制。电镜可见核质比高、细胞器不发达,具干细胞特征的细胞。诱导后神经干细胞与诱导前相比,细胞分化以及降解有关的细胞器(如溶酶体等)、自噬小体显著减少。流式细胞仪检测诱导前后的神经干细胞球中的SP细胞,分别为10.43%、16.89%,所有细胞均为二倍体,大部分细胞位于G0/G1期。免疫荧光显示,未诱导神经干细胞球体大部分细胞表达nestin,分化细胞分别表达GFAP和MAP2。诱导后的神经干细胞球体中,大部分细胞表达nestin,少部分表达分化标志物GFAP、NSE,而部分分化细胞仍然共表达干细胞标志物nestin和分化标志物GFAP和MAP2,甚至同时表达两种分化标志物GFAP和MAP2。(3)肿瘤细胞在无血清培养条件下,呈悬浮的球状生长,在含血清培养条件下贴壁并分化为瘤性胶质细胞、神经元。电镜下见到核质比高、细胞器不发达、与细胞分化降解有关的细胞器罕见。流式细胞仪检测肿瘤细胞球中的SP细胞为17.27%,异倍体细胞为26.77%,大部分细胞处于S期。免疫荧光染色发现,细胞球中大部分细胞表达干细胞标志物nestin,少数细胞表达分化标志物GFAP和NSE。贴壁细胞大部分仍然共表达干细胞标志物nestin和分化标志物GFAP和MAP2,甚至同时表达两种分化标志物GFAP和MAP2。脑肿瘤干细胞和诱导后,神经干细胞高表达c-myc, MDM2(p53抑制基因)结论：(1)成功建立更加稳定的、可调控的、具有干细胞特性的脑肿瘤小鼠模型,为进一步研究脑肿瘤干细胞的特性奠定基础。(2)成功从诱导前后的Tet-on转基因成体小鼠室管膜下区中,分离到神经干细胞,且发现诱导后的神经干细胞表现为增殖活性增加和分化抑制,表明神经干细胞已经出现早期恶性转化表现。(3) Tet-on转基因小鼠脑肿瘤组织中,存在具有自我更新和多向分化潜能的脑肿瘤干细胞,且发现脑肿瘤干细胞与诱导后的神经干细胞一样,表现为增殖活性增加和分化抑制。C-myc通路活化和p53通路破坏,在脑肿瘤干细胞维系与神经干细胞恶性转化中起关键作用。(4)在同一动物模型个体中,同时分离到脑肿瘤干细胞和神经干细胞,使该模型成为肿瘤干细胞研究的有力工具。更多还原

【Abstract】 Objective:To establish stable regulated brain tumor mouse model with sternness coexpressing c-myc and SV40Tag induced by Tet-on system and lay the ground work for future research on the special property of the brain tumor stem cells (BTSCs). To isolate and identify normal adult neural stem cells (NSCs), induced adult neural stem cells (NSC’s) and mouse brain tumor stem cells from subventricular zone (SVZ) tissue of non-induced genetically engineered mouse models, SVZ tissue and tumor tissue of induced genetically engineered brain cancer mouse models respectively. To learn the similarities and differences among normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells, and define the differential molecular mechanisms. To provide evidences for the hypothesis that brain tumor stem cells originate from neural stem cells.Methods:Transgenic mouse coexpressing pTRE2-c-myc, Tet-on and pTRE2-SV40Tag were generated by crossing the pTRE2-c-myc transgenic mouse with transgenic mouse coexpressing both Tet-on and pTRE2-SV40Tag, and induced by doxycycline (DOX) to generate brain tumor mouse model. Magnetic resonance image, macroscopy and histopathology of the tumor specimen and immunohistochemistry were used to investigate the characteristics of the model. Cells from SVZ tissue of non-induced genetically engineered mouse models and SVZ tissue of induced genetically engineered brain cancer mouse models were cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate. Differentiated cells of the two group cell spheres were cultured in DMEM/F12 containing 10% FCS. Brain tumor tissues were obtained from genetically engineered mouse which symptoms appear. Cells from tumor tissue were cultured in DMEM/F12 mediume containing fibroblast grwoth factor-2 (bFGF), epidermal growth factor (EGF) and B27 minus retinyl acetate. Differentiated cells of mouse brain tumor spheres were cultured in DMEM/F12 containing 10% FCS. The morphous and proliferation of normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells were obeserved under phase contrast microscope. The ultrastructures of the three group cells spheres were obeserved under transmission electron microscope (TEM). To determine whether the three group cells spheres contain stem cells, Hoechst 33342 was used to sort for the side population (SP) phenotype. Cell cycle and chromosome ploidy of the three group cells spheres were performed on Flow cytometry. Multiple immunofluorescence was used to assays the cell markers of the three group cells spheres and differentiated cells. Immunofluorescence was used to define the differential expression in c-myc and p53 pathway among normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells.Results:(1) Thirty of Seventy-three transgenic mouse coexpressing pTRE2-c-myc, Tet-on and pTRE2-SV40Tag generate brain tumor induced by DOX. The incidence of brain tumor was 41.1%. Beside of one brain stem tumor, all of the other tumors were located on the pineal region. On magnetic resonance imaging (MRI), these lesions appear isointense to hyperintense on TI-weighted images and and isointense to hypointense on T2 weighted images. These lesions were enhanced and rather homogeneous to moderately heterogeneous with contrast images. The brain tumors were diagnosed as undifferentiated medulloblastoma. The brain tumors were highly invasive. And immunohistochemistry indicated that the tumors overexpressed c-myc, SV40Tag and stem/progenitor cells mark nestin.(2) Cells from SVZ of uninduced and induced genetically engineered adult mouse formed cell spheres when cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate. Uninduced adult neural stem cells spheres differentiated into neuronal and glial cells when cultured in DMEM/F12 containing 10% FCS. Induced adult neural stem cell spheres exhibited enhanced proliferating capacity and impaired differentiation. The ultrastructures of uninduced and induced adult neural stem cell spheres indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. Compared with uninduced adult neural stem cell spheres, cellular organs associated with differentiation and degradation, for example lysosome, autophagosome, were significant reduced in induced adult neural stem cell spheres. The average first sort percentage of SP cells was 10.43% and 16.89% in uninduced and induced adult neural stem cell spheres respectively. All uninduced and induced adult neural stem cell spheres were diploid. A majority of cells in uninduced adult neural stem cell spheres were at S phase. A majority of cells in uninduced adult neural stem cell spheres were nestin positive. The differentiated cells of uninduced adult neural stem cell spheres were GFAP and MAP2 positive respectively. A majority of cells in induced adult neural stem cell spheres were nestin positive, and small fraction were GFAP and NSE positive. But partially differentiated cells still coexpressed stem cells marker nestin and differentiation markers GFAP and MAP2, and even coexpressed differentiation markers GFAP and MAP2.(3) Cells from genetically engineered mouse brain cancer tissue formed stem-like sphere when cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate, and differentiated into neuronal and glial like cells when cultured in DMEM/F12 containing 10% FCS. The ultrastructures of brain tumor sphere indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. Cellular organs associated with differentiation and degradation were seldom seen. The average first sort percentage of SP cells was 17.27%. Heteroploid in mouse brain cancer spheres was 26.77%. A majority of cells were at S phase. A majority of cells in mouse brain cancer spheres were nestin positive and small fraction of cells were NSE and GFAP positive. Most differentiated cells coexpress nestin with MAP2 or GFAP, and even coexpressed differentiation markers GFAP and MAP2. Brain tumor stem cells and induced neural stem cells overexpressed c-myc and MDM2 (negative regulator of p53).Conclusion:(1) Stable regulated brain tumor mouse model coexpressing c-myc and SV40Tag induced by Tet-on system was established. The ground work was laied for future research on the special property of the brain tumor stem cells (BTSCs).(2) Adult neural stem cells were isolated from SVZ of uninduced and induced Tet-on genetically engineered mouse models. Induced neural stem cells exhibited enhanced proliferating capacity and impaired differentiation. Our data indicated adult neural stem cells acquired early malignant transformation.(3) Tet-on genetically engineered mouse brain cancer contains brain tumor stem cells which have the capacity to self-renew and muti-differential potentiality. Brain tumor stem cells exhibited enhanced proliferating capacity and impaired differentiation as same as induced neural stem cells. Activation of c-myc and inactivation of p53 pathway play critical roles in substance of brain tumor stem cells and malignant transformation of neural stem cells.(4) Adult neural stem cells and brain tumor stem cells were isolated from one Tet-on genetically engineered brain cancer mouse models. Tet-on genetically engineered brain cancer mouse models become powerful tool for brain tumor stem cells research.更多还原