【作者】 王婷；

【导师】 王百忍；

【作者基本信息】 第四军医大学 ， 神经生物学， 2008， 博士

【摘要】 长期以来脑膜被视为是仅具有保护作用的非神经组织,在结构和功能上与脑实质有本质的不同,但在上世纪90年代Mercier等应用形态学方法清晰地勾勒出柔脑膜细胞与脑实质内胶质细胞、神经元之间有网状的复杂联系,从而提出柔脑膜细胞参与脑内某些生理或病理过程的可能性,引发了对柔脑膜功能的深入思考。新近的一些研究发现:脑膜中的柔脑膜细胞(leptomeningeal cells)表达和分泌多种细胞因子和生物活性物质,参与中枢的免疫调节和诱导发育神经元的迁移定位,并与小脑功能的完善及脑内酶屏障的构成密切相关。但是,柔脑膜细胞与神经元生长、分化的关系还存在争议。有研究认为柔脑膜细胞分泌的抑制神经突起生长的因子是神经元损伤后修复的障碍;也有研究指出柔脑膜细胞能分泌神经生长因子等多种促神经突起再生的因子,并作为滋养层支持神经干细胞的体外生长。新近的研究还注意到成年大鼠柔脑膜层有神经干细胞标记物Lex/ssea-1的表达。这些结果提示柔脑膜细胞与神经系统损伤修复、尤其与神经干细胞之间可能存在着某种特殊联系,为进一步探索柔脑膜细胞的功能提供了新思路。针对以上问题,我们以柔脑膜细胞为研究对象,设计了五组实验,从在体和体外细胞培养的角度,结合应用柔脑膜铺片、细胞培养、免疫组织化学染色、免疫荧光标记、BrdU掺入实验、Western blot等研究方法,对柔脑膜细胞可能具有的神经干细胞特性做了初步探讨。第一组实验观察了正常成年以及不同生长发育期大鼠脑内柔脑膜细胞是否表达经典的神经干细胞标志巢蛋白(nestin);第二组实验在成功建立单纯柔脑膜细胞培养体系的基础上,检测了柔脑膜细胞能否表现出自我更新以及多向分化等神经干细胞的生物特性;第三组至第五组实验分别观察了不同调控因素(白细胞介素-6、抗抑郁药物以及机械损伤)对于柔脑膜来源神经干细胞增殖分化的影响。主要结果有:实验一一、正常成年SD大鼠及Balb/c小鼠的柔脑膜层中均可检测到神经干细胞特异性标记物nestin的表达,但表达水平低,主要局限于脑腹侧及大脑镰两侧的脑膜组织。两种动物柔脑膜组织中阳性细胞的形态与分布存在差异。二、不同生长发育期SD大鼠的柔脑膜细胞中nestin蛋白表达水平随生长发育呈进行性降低。柔脑膜铺片中除nestin/Thy1.1双标阳性的柔脑膜细胞外,也有GFAP/nestin阳性细胞存在。以上结果说明,不同生长发育期SD大鼠的柔脑膜细胞均可表达神经干细胞标志nestin,即具有神经前体细胞的增殖潜能,但此潜能随生长发育而进行性减弱。实验二一、比较脑膜组织在不同培养条件下柔脑膜细胞的纯度,发现以DMEM/5%FCS/5%HS培养时,培养体系中除柔脑膜细胞外,还存在约30%的星形胶质细胞和少量神经元;以DMEM/1%FCS/9%HS培养时,柔脑膜细胞纯度达90%以上,几乎没有神经元沾染。后者继续传代可使其纯度达97%以上,仅有极少数星形胶质细胞存活。结果提示改变培养条件及多次传代是建立单纯柔脑膜细胞体系的有效方法。二、检测单纯培养的柔脑膜细胞是否具有神经干细胞的基本生物学特性。发现:给予含生长因子EGF/bFGF的无血清条件性培养基后,柔脑膜细胞可自我增殖形成神经球样结构,其成球性呈bFGF浓度依赖性;再以胎牛血清诱导分化,可见除Thy1.1阳性的柔脑膜细胞外,还可分化为β-tubulin III、GFAP、RIP阳性的神经元及胶质细胞;并且柔脑膜细胞经多次传代仍可形成克隆球及多向分化,但能力逐渐减弱。以上结果说明,间充质来源的柔脑膜细胞经体外分离培养后能够表现出自我更新以及多向分化等神经干细胞的基本生物学特性,并可分化形成神经元。实验三一、白细胞介素-6(IL-6)可以诱导柔脑膜来源的神经球向神经元及胶质细胞多向分化,并显著提高分化神经元的比例。以多种神经元标记物鉴定分化神经元性质,提示其为5-HT阳性的未成熟神经元。二、免疫细胞化学染色及Western blotting结果均提示,IL-6孵育可显著上调其自身受体(IL-6Rα及gp130)在柔脑膜来源神经球内的表达,为柔脑膜细胞感受IL-6信号并发生应答反应提供了形态学基础。三、上述体系中,IL-6也引起磷酸化ERK1/2及STAT3分子在柔脑膜来源神经球中的表达。而分别给予其特异性抑制剂PD98059及AG490后,发现ERK1/2通路磷酸化的抑制可影响神经球向神经元及胶质细胞的分化,而STAT3通路磷酸化的抑制则以星形胶质细胞分化的受抑为主。同时, STAT3分子磷酸化提示gp130处于活化状态。以上结果说明,细胞因子IL-6可促进柔脑膜来源神经干细胞向神经元分化,而分化神经元呈5-HT反应阳性。IL-6刺激引起了其自身受体及磷酸化ERK1/2及STAT3信号分子在该细胞体系中的表达,提示上述细胞内分子机制可能参与了柔脑膜细胞向神经元、胶质细胞的分化。实验四一、以抗抑郁药物西酞普兰孵育柔脑膜来源的神经球,发现:西酞普兰能够维持神经球的克隆化生长、促进细胞增殖,并引起细胞内ERK1/2、STAT3信号分子磷酸化;以信号通路抑制剂预处理的结果显示,西酞普兰诱导细胞增殖的效应表现为ERK1/2通路依赖性。二、观察西酞普兰孵育对柔脑膜来源的已贴壁分化的神经干细胞的影响,发现:西酞普兰可促进神经干细胞继续分化,并显著上调分化神经元的比例;同时也引起了分化细胞内磷酸化ERK1/2、STAT3分子的表达。以信号通路抑制剂预处理的结果显示,上述体系中神经元和胶质细胞的分化分别表现为ERK1/2或STAT3通路依赖性。以上结果说明,西酞普兰可以诱导柔脑膜来源神经干细胞的增殖,也可促进其向神经元分化。但是调控细胞增殖分化的机制不同,分别表现为ERK1/2通路或ERK1/2及STAT3通路依赖性。实验五一、建立柔脑膜细胞的体外机械损伤模型,检测机械损伤对细胞增殖及逆分化的影响。结果显示:划伤刺激促进了柔脑膜细胞增殖,但不能诱导其表达nestin蛋白;而含生长因子的无血清条件性培养基可诱导损伤细胞形成神经球,后者在血清存在时向神经元及胶质细胞分化。二、观察损伤的柔脑膜细胞对正常星形胶质细胞增殖及逆分化的影响,发现:损伤的柔脑膜细胞可以诱导正常星形胶质细胞增殖并表达nestin,后者也可形成神经球并向神经元、胶质细胞分化。以上结果说明,机械损伤虽然不能直接诱导柔脑膜细胞分化为神经前体细胞,但可以刺激细胞分泌某些活性物质,诱导与之共培养的星形胶质细胞增殖并逆分化为神经前体细胞。并且,细胞微环境对于细胞能否表现出神经干细胞特性具有重要意义。结论本研究从不同的角度证实:起源于间充质组织的柔脑膜细胞能够表现出神经干细胞的基本特性,自我增殖并在适当条件下分化为神经元、星形胶质细胞和少突胶质细胞;不同性质的刺激(细胞因子、抗抑郁药物以及机械损伤等)可调控其增殖分化;神经系统损伤时,柔脑膜细胞可能分泌活性物质而参与诱导脑内内源性神经干细胞的增殖活化。根据以上结果,我们提出了脑膜细胞可能是脑内潜在的神经干细胞的观点。这些初步发现将为我们更加全面、深入地认识柔脑膜细胞的功能奠定了基础,为神经损伤后的干细胞修复提供了新的思路。更多还原

【Abstract】 Traditionally, meninges have been thought to be only a protective apparatus and structurally and functionally separated from brain parenchyma. In 1990s, Mercier et al challenged this prevailing view by using immunohistochemical methods to vividly mark the cellular network formed by leptomeningeal cells, glial cells and neurons. This conception of meningeo-glial network calls for a reconsideration of the role of meninges and appears to reflect the possibility of leptomeningeal cells’involvement in physiological or pathological processes in CNS. Increasing evidences show that meninges are composed by various cells besides fibroblasts, which facilitate their complicated functions. Leptomeningeal cell, a special kind of fibroblast-like cell in CNS, has become a hot topic. For example, it is recognized that leptomeningeal cells can express and secret various cytokines and biological molecules, participate in the neuroimmunomodulation in CNS, be actively involved in neuronal cells differentiation and migration, and play key role in cerebellum maturation and enzyme-barrier constitution.However, the role of leptomeningeal cells in inducing neuronal growth and differentiation is still unclear. Some researchers held the opinion that leptomeningeal cells produced inhibiting molecules to form the barriers of neural regeneration. In contrast, other evidences demonstrated that leptomeningeal cells mainly promote the neurites growth by secreting neurotrophic factors, such as neural growth factors, and served as“feeder-layers”to sustain the proliferation of neural stem cells. Recently, it was showed that Lex/ssea-1, a specific marker for neural stem cell, was also expressed by cells in leptomeninges in adult rats. All of these results indicate that leptomeningeal cells might play special roles in neurogenesis and interact with neural stem cells, unveiling the study of leptomeningeal cells’characteristics and novel potentials.Accordingly, aiming to elucidate the possible identity of leptomeningeal cells as neural stem cells, we designed five sets of experiments in this study, which were made on animal models and cultured cells, respectively. The methods used were leptomeningeal wholemount preparation, cell culture, immunohistochemistry, immunofluorescence, BrdU incorporation and Western blotting analysis. In the first part of study, we detected the expression of nestin protein, a specific marker for neural stem cell, by the leptomeningeal cells in rats of different developmental stages. In the second part of study, after establishment of a relatively pure culture system of leptomeningeal cells, we detected whether these cells could exhibit self-renewal and differentiation potentials. Then in the following experiments, we observed the effects of three kinds of regulation factors, which included interleukin-6, anti-depression drugs and mechanical injury respectively, on the proliferation and differentiation of leptomeninges-derived neural stem cells.The main results are as follows:Part One 1.It was showed that leptomeningeal cells in adult SD rats and adult Balb/c mice could express nestin, a specific marker for neural stem cell. However, the expression level was quite low and the positive cells were only limited in special location of leptomeninges, mainly referred to which overlaid on the ventral brain and cerebral falx. In addition, the morphology and the distribution of positive cells in leptomeninges were different between two species of animals.2.It was detected that leptomeningeal cells expressed nestin in rats at different developmental stages and the expression level diminished gradually as development advancing. Moreover, we found that cells in wholemount preparation were not only double-immunostained by nestin/Thy1.1, but also by GFAP/nestin.These results indicate that nestin, a specific marker for neural stem cells, could be expressed by leptomeningeal cells in rats of different developmental stages. That is to say, leptomeningeal cells possessed stem cell-like proliferation potential, although this feature was diminished as the development advancingPart Two1.After leptomeningeal cells were cultured in different condition, we compared the purity and cellular property in these culture systems. When cultured in DMEM containing 5% FCS and 5% HS, leptomeningeal cells were mixed with 30% of astrocytes and few neurons, But in DMEM/1%FCS/9%HS, the leptomeningeal cells accounted for more than 90% of the total cells and glia and neurons were rarely seen. In addition, sequential passages of the latter system improved the cellular purity to more than 97% and only a few astrocytes were found in this culture system. Therefore, changing media and passage during culture processes were effective to establish a pure culture system for leptomeningeal cells.2.Accordingly, we detected whether cultured leptomeningeal cells could exhibit essential characteristics of neural stem cell. The results showed that in the presence of EGF/bFGF containing serum-free conditioned medium, leptomeningeal cells proliferated to develop into neurosphere-like morphology. This process was in a bFGF-dependent manner. Then serum administration induced these leptomeningeal cells to differentiate into the neuronal and glial lineage cells, marked byβ-tubulinIII, GFAP and RIP respectively. Moreover, it was showed that after repetitious passages, leptomeningeal cells still generated neurospheres and multipotentially differentiated, though such capacity declined gradually.These results indicate that the mesenchyma-derived leptomeningeal cells possess the capacity of neural stem cell to self-renew and differentiate multipotentially, especially producing neurons.Part Three1.It was revealed that Interleukin-6 administration not only led the leptomeninges-derived neurospheres to generate neurons and glial cells, but also elevated the percentage of neuronal lineage cells. Then using different neuronal markers, these newly formed neurons were found to be 5-HT positive immature neurons.2.It was revealed by the immunocytochemical and Western blotting results that IL-6 administration up-regulated the expression of its own receptor(sIL-6Rαand gp130)in these leptomeninges-derived neurospheres. In this regard, the presence of IL-6 receptors enabled cells to sense and react to IL-6 stimulation. 3.In above system, IL-6 also induced the phosphorylation of ERK1/2 and STAT3 molecules in the cells. Then we administrated pharmacological inhibitors individually before IL-6 incubation, as PD98059 and AG490. It was revealed that PD98059 pre-treatment decreased the generation of neurons and glial cells, while AG490 administration only down-regulated the GFAP level. In addition, the phosphorylation of STAT3 molecules also indicated the activation of gp130.These data suggest that IL-6 could promote the differentiation of leptomeninges-derived neural stem cell into neurons, which were mainly serotonergic immature neurons. In addition, IL-6 administration increases the expression of its own receptors and the phosphorylation of signal molecules, ERK1/2 and STAT3, in these cells. Thus, these molecules may cooperatively involve in the differentiation of leptomeninges-derived neurospheres induced by IL-6 stimulation.Part Four1 . After incubated the leptomeninges-derived neurospheres with citalopram, one kind of anti-depression drugs, we detected that citalopram not only supported the clonal morphology and the proliferation of cells, but also induced the phosphorylation of ERK1/2 and STAT3 signal molecules. Moreover, using pharmacological inhibitors of these two molecules further indicated an ERK1/2-dependent effect of citalopram on the cellular proliferation.2.On the other hand, we also detected an effect of citalopram on the differentiation potentials of leptomeninges-derived neural stem cells. It was showed that when the neurospheres had attached to the coverslip and tended to differentiate, citalopram administration could not only significantly promote their differentiation processes, but also improve the neurogenesis. Similarly, it induced the phosphorylation of ERK1/2 and STAT3 in these cells, while the gliogenesis and neurogenesis exhibited these two pathways related manner respectively.These data suggest that citalopram could promote the proliferation and neurogenesis of leptomeninges-derived neural stem cells. But the regulation mechanisms are quite different. In other word, the cellular proliferation involves ERK1/2 pathways only, while the differentiation is dependent on both pathways.Part Five1.We established a leptomeningeal cells’mechanical injury model and examined the capacity of these cells to proliferate or de-differentiate. It was revealed that, though the injury promoted the proliferation of cells, the expression of nestin couldn’t be detected simultaneously in these cells. However, in this injury model, cells were found to be able to generate neurospheres in the presence of growth factors containing serum-free medium, and differentiate into neuronal or glial lineage cells when re-incubated with serum.2.To explore the effects of injured leptomeningeal cells on the normal astrocytes, we co-cultured these two cells to detect the latter cells’capacity of proliferation and de-differentiation. It was showed that normal astrocytes could proliferate and express nestin protein in the presence of injured leptomeningeal cells. In addition, astrocytes in this system could also develop into neurospheres and then generate neurons or glial cells when cultured in serum-free or serum containing medium respectively.These results indicate that though the mechanical injury couldn’t induce the leptomeningeal cells to de-differentiate into neural progenitors directly, it increases the production of certain diffusible factors by the cells which then induced the de-differentiation and proliferation of astrocytes. Moreover, the environmental factors play key roles in directing cells to exhibit neural stem cell-like potentials.ConclusionIt can be verified by the present study that the mesenchyma-derived leptomeningeal cells possess the essential characteristics of neural stem cells. They can self-renew and proliferate, and then differentiate mutipotentially into neuronal and glial lineage cells under certain circumstances. In addition, different kinds of stimuli, such as cytokines, anti-depression drugs and mechanical injury, can involve in their proliferation or differentiation processes. Also, injury may lead the leptomeningeal cells to secret some factors to induce the activation and proliferation of neural stem cells in situ in CNS. In a word, it is helpful for us to establish a comprehensive and in-depth understanding of leptomeningeal cells’functions, which will well ground their clinical application.更多还原