【作者】 姬西团；

【导师】 章翔；

【作者基本信息】 第四军医大学 ， 外科学， 2004， 博士

【摘要】 神经干细胞（neural stem cells，NSCs）是具有趋向性、自我更新和多向分化潜能的特殊细胞。应用NSCs特性，治疗神经系统多种疾病具有广泛的前景，尤其在神经退行性疾病、缺血性疾病的功能替代以及作为基因载体治疗胶质瘤等方面更是如此。到目前为止，NSCs的一些特性仍然不清。有学者认为NSCs分化与细胞的内环境、神经营养因子、分化因子、细胞的信号转导等有关。然而，由于不同来源的NSCs对同一分化因子反应各异，同一来源的NSCs对不同分化因子或不同浓度亦反应不同，致使这一研究结果莫衷一是。另外，克隆球内及其之间能否形成突触联系，以及细胞连接等信息传递的形态学结构，目前尚未见到有关报道。为此，本研究将分为四大部分对NSCs的生物学特性进一步进行研究与探讨。 实验一 人、鼠神经干细胞体外培养及鉴定 （1）人NSCs的培养及鉴定迅速取出10～14w人胚胎脑海马区组织，将其置入无血清的DMEM培养液中初步处理后，用含人源性表皮生长因子（Human epidermal growth factor，HuEGF）、碱性成纤维生长因子（Fibroblast growth factor，basic fragment，bFGF），改良的Eagle DMEM培养基(Dulbecco Eagle’s minimum essential medium DMEM／F₁₂)中进行培养。脑海马区组织富含有干细胞特性的细胞，该细胞在无血清的条件培养基中能持续增殖，并有形成细胞克隆的能力。随后借用免疫组织化学的方法对所培养的细胞进行鉴定，结果证明，经过数天培养的原代细胞巢蛋白（nestin）抗原呈阳性反应。培养细胞团在含血清的培养基中经诱导分化后，成熟分化为神经细胞，部分呈神经丝-200（Neurofilament-200，NF-200）抗原阳性，部分呈胶质纤维酸性蛋白（glial fibrillary 第四军医大学博士学位论文acidicProtein，GFAP）抗原阳性。说明原代培养细胞在血清中能分化为神经元及神经胶质细胞。这些均证明所培养的细胞系NSCs。 （2）小鼠NSCs的培养与鉴定在无菌条件下剖腹取胎鼠，胎头放入无血清DMEM培养基的培养皿中。随后分离胎鼠大脑皮质，用含鼠源性表皮生长因子（叩idermal盯ov沈h factor from mouse，EGF）及碱性成纤维生长因子（bFGF）的DMEM条件培养基进行培养。由于胚小鼠脑皮层中可以分离出有干细胞特性的细胞，该细胞在无血清的条件培养基中能持续增殖，并有形成细胞克隆的能力。随后借用免疫组织化学的方法对所培养的细胞进行鉴定。这些原代培养细胞nestin抗原呈阳性。在含血清的培养基中，这些细胞能分化为成熟的神经细胞。分化细胞中部分呈NF一200抗原阳性，部分呈GEAP抗原阳性。说明原代培养细胞在血清中能分化为神经元及神经胶质细胞。这些均证明所培养的细胞系NSCs。实验二外来信号对神经干细胞定向分化的调控 目前的研究认为，决定NSCS定向分化的机制有两种:一种是细胞自身基因调控，另一种是外来信号调控。本实验通过改变NSCs培养条件，观察其对NSCs定向分化的影响。结果显示:（l）单纯用含表皮生长因子（EGF）的DM卫M培养液培养NSCs，小鼠NSCs生长良好，在加血清诱导分化后，则主要为星形胶质细胞，仅有个别的神经元。而人胚胎NSCs除克隆球较小鼠的形成慢且较松散外，其余基本相同。（2）用含碱性成纤维生长因子伪FGF)的DMEM培养液培养NSCs，小鼠与人NSCs均生长良好。然而，在加血清诱导分化后，不同浓度的bFGF培养的Nscs其分化的细胞不同。当浓度为1 ong/ml时，分化细胞以神经元为主。而当bFGF浓度为加n岁ml时，分化为胶质细胞的比例明显增加，同时神经元明显减少。（3）用同时含碱性成纤维生长因子伪FGF)与表皮生长因子（EGF）的DMEM培养液培养NSCs，小鼠与人NSCs生长及神经球的形成良好。在加血清诱导分化后，含两种促进NSCs分裂因子培养的NSCs其分化的细胞更接近体内NSCs分化的比例，其神经元约为35%，胶质细胞约为55%，其余为小星形胶质细胞。当两种因子浓度同时增加或减少时（10ng/ml²0ng/Inl），分化细胞的比例无明显变化。（4）神经生长因子（nery。脚认吐伪etor，NoF）对Nses的影响。研究结果表明，NGF对神经球的形成无明显的影响，但可促使其向神经元分化。（5）维甲酸（retinoic aeid，RA）对NSCs分化的影响，RA可能在神 第四军医大学博士学位论文经再生和功能重建方面发挥重要作用。本实验对体外培养NSCs加入RA，观察对NSCs的定向分化影响。结果见神经克隆球形成缓慢且较少，而直接分化为神经元的细胞比例明显增加。这将为神经细胞脑内移植提供充足和理想的材料。（6）分别用条件培养基（同时含EGF与bFGF的DMEM培养液）及IOmLL一，20mLL一l、30mLL一’、40mLL一‘、somLL一’、100mLL一’胎牛血清+条件培养基，对人胚海马区及小鼠胚胎脑组织进行分离、培养，用免疫组织化学鉴定。随后对不同培养条件下、不同生长时期NSCs生物学特性进行光镜观察。结果显示，1 omLL一’、20mLL·L一‘胎牛血清+条件培养基组培养的Nscs比单独条件培养基组克隆球多而大。30mLL一，、40mL.L一’、50mLL一’组既有部分贴壁细胞，也有大量的克隆球形成，克隆球有伪足样突起，克更多还原

【Abstract】 Neural stem cells (NSCs) are special cells which have some capacity of tropism for tumors, self-renewal, and the potencies to differentiate with multi-direction. There is a broad prospect to use the property of neural stem cells for some diseases treatment in central nervous system, especially, in cell substitute therapy, gene carrier for gene therapy, treatment of CNS trauma, degeneration diseases of CNS and brain tumor, etc. Additionally, the discovery of neural stem cells is a breakthrough for neural regeneration in the central nervous system (CNS). Up to now, some of the properties on neural stem cells are not still clear. Some scholars considered that differentiation of neural stem cells are relevant to the internal environment of cells, neurotrophic factor, differentiating factors and cellular signal transduction. However, the neural stem cells from various resources of species show different reactive capacity to the same differentiating factor. Even so, neural stem cells from the isotype species demonstrate various response to the different factors and different concentration of facilitating differentiating factors. With the exception of above-mentioned, the morphologic structure on biological signal transmission of NSCs, structure of junction-like among neurospheres was not reported in the past papers and webs. To explore the biological property of neural stem cells (NSCs) from embryonic mouse cerebral cortex and fetal hippocampus comparatively and study the molecular mechanism of development of nervous system, we did the study as following.Section oneCultivation, identification of neural stem cells from both embryonic mousecortex and human fetal hippocampus in vitro(1) Cultivation, identification of neural stem cells from human fetal hippocampus in vitro The first step in this part was to dissect and separate hippocampus from the embryo-brain of 10-14weeks. Then, the human stem cells were, isolated from the tissue selected in medium containing HuEGF and bFGF. The cells from fetal hippacampus had an ability of consecutivedifferentiation and forming neurospheres in serum-free medium. Both clusters of cells and naive cells were positive by nestin immunocytochemical staining. Neurofilament-200(NF-200) and glial fibrillary acidic protein (GFAP) antigens were positive in the mature cells, which demonstrated that some of the differentiated cells were neurons and the others were glial cells. These results showed that the primary culture cells were NSCs.(2) Cultivation, identification of neural stem cells from the cerebral cortex of embryo mouse.In this section, the conceived murine belly was cut open under aseptic condition firstly. The next was to isolate the stem cells from the mice cortex in serum-free medium containing EGF from the mouse and bFGF. The primary culture cells had the ability to consecutively proliferate and form neurosphere in the medium. Nestin was positively expressing in these cells which were examined by immunohistochemistry. The cells could differentiate into adult neural cells in medium supplemented with fetal bovine serum. The expression of NF-200 was positive in some of the differentiated cells and some of them were GAFP positive. It demonstrated that some of the differentiated cells were neurons and the others were glial cells. These results showed that the primary culture cells were NSCs.Section twoEffect on regulation of NSCs orientating differentiation by ecdemic signalBy now, there are two kinds of mechanisms on orientating differentiating theory of neural stem cells. One is NSCs autogene regulation and the other is exotic signal regulation. This experiment was designed to explore the morphologic structure on biological signal transmission of neural stem cells (NSCs), morphologic characteristics of NSCs cultured in vitro under the different medium. The results showed as follows. (1) The neural stem cells from mice grew very well in the medium with EGF only. However, most neural stem cells could be induced to differentiate into glial cells and only a few neurons when更多还原