论文标题:人胚脑组织神经干细胞的分离培养与鉴定
Isolation, Culture and Identification of Neural Stem Cells of Human Embryonic Brain Tissue
论文作者 赵新利
论文导师 杨波；宋来君；杜英,论文学位 硕士,论文专业 神经外科
论文单位 郑州大学,点击次数 70,论文页数 55页File Size2423k
2003-06-02论文网 http://www.lw23.com/lunwen_68322397/ 神经干细胞;神经上皮干细胞蛋白;免疫细胞化学;白血病抑制因子;单唾液酸四己糖神经节苷脂
neural stem cells;Nestin;immunocytochemistry;LIF;GM-1
神经干细胞(Neural stem cells，NSCs)是一种终生具有自我更新能力的细胞，可增殖分化产生中枢神经系统的3种基本细胞：神经元、星形胶质细胞和少突胶质细胞。NSCs的发现和深入研究可能为治疗神经系统疾病带来新的契机。但是，在中枢神经系统中，发育阶段不同、部位不同，NSCs所占的比例不同，且普遍比例较低。自NSCs被发现以来，建立体外培养体系，用不同的因素刺激NSCs快速增殖，获取大量的NSCs，寻找影响NSCs分化的不同因素，提高神经元在分化中所占的比例，是国内外许多学者一直研究的方向和目标。白血病抑制因子(Leukemia inhibitory factor，LIF)是一种多功能的糖蛋白，其分子量为20kD，含有180个氨基酸残基，它能够诱导巨细胞的分化并抑制鼠M1骨髓瘤细胞的增殖。LIF与IL-6(Interleukin-6)、G-CSF(Granulocyte-colony stimulating factor)一起在造血干细胞(Hematopoietic stem cell)的早期调控中具有重要作用。神经节苷脂(Gangliosides)是细胞膜的重要组成成分，在中枢神经系统中含量较高。神经节苷脂与多肽生长因子一样，被认为是与神经细胞发育、分化、存活及病理的所有阶段相关联的营养单位。由于它们调控过程复杂，它们在许多方面的生理角色尚未被揭示。单唾液酸四己糖神经节苷脂(Monosialotetrahex-osylganglioside，GM-1)是哺乳动物神经节苷脂的主要种类，外源性GM-1能够通过血脑屏障，嵌入损伤的神经细胞膜，具有明显的抗神经细胞损伤、促修复和促神经细胞生长的作用。近年来，国内外有关NSCs的研究，多数停留在鼠的NSCs，对人胚NSCs研究相对较少，LIF对NSCs增殖郑州大学2003届硕士研究生毕业论文人胚脑组织神经干细胞的分离培养与鉴定和分化的影响，国外学者做了一些研究工作，而国内文献报道较少，GM一1对人胚中枢神经系统NSCs的作用更为鲜见。本实验，机械分离10周人胚脑室区/脑室下区(V entricular zone/subventricular zone，Vz/Svz)组织，用无血清培养基(各组添加成分不同)对NSCs进行培养，比较不同培养条件下细胞生长情况;在相同或不同条件下诱导细胞分化，比较分化后神经元及神经胶质细胞所占比例;探讨了机械分离法对NSCs培养的适用性和LIF、GM一1对NSCs生长和分化的影响。用免疫细胞化学法检测培养细胞的神经上皮干细胞蛋白困euroePithelial stem cen Protein，Nestin)和增殖细胞核抗原(Proliferating eell nuelear antigen，pCNA)及分化后细胞的神经元特异性烯醇化酶(Neuronspeeifie enolase，NSE)或胶质纤维酸蛋白(Glial fibrill娜acidie拼otein，GFAp)，对NSCs进行了鉴定。 方法:取材于10周人胚VZ/SVZ组织，使用机械分离法分散脑细胞，按基础培养基(DMEM邝12+BZ:)中添加成分的不同，分五组:A组加入bFGF(20ng/ml)，B组加入bFGF(2 ong加l)+EGF(20ng/ml)，C组加入bFGF(20ng/ml)+EGF(ZOng/ml)+LIF(1 on留ml)，n组加入bFGF(Zong/ml)+EGF(Zong/inl)+GM一1(33.5n留ml)，E组为GM一l(33.5ng/ml)，每组两瓶，置入COZ培养箱(5%C02，37℃)进行培养。①自A组、B组、C组中分别取少量细胞悬液，均调制成细胞密度为1 xl护个/m1，接种于%孔培养板，每组12孔，每孔0.lml，置入CO:培养箱培养。于培养的4d、sd用MTT比色法检测各组OD值，了解LIF对NSCs增殖的影响。②自B组、D组、E组中分别取少量细胞悬液，另增加F组(含基础培养基，无添加成分)作为阴性对照，四组均调制成细胞密度为IX10，个/ml，接种于96孔培养板，每组12孔，每孔0.lml，置入eoZ培养箱培养。于培养的4d、8d用MTT比色法检测各组OD值，了解GM一1对NSCs增殖的影响。③对A、B、C、D、E五组来源的NSCs在相同的条件下进行诱导分化或④对同一培养来源(B组)的NSCs在不同的条件( bFGF、LIF、GMI三个浓度、5%胎牛血清)下诱导分化，用免疫细胞化学方法检测NSE和GFAP表达阳性率，了解LIF、GM一1对NSCs分化方向的影响。⑤对培养的细胞用免疫细胞化学方法检测Nestin和PCNA，结合细胞培养传郑州大学2003届硕士研究生毕业论文代和分化情况，鉴定NSCs。人胚脑组织神经干细胞的分离培养与鉴定 结果:①LIF对NSCs增殖的影响，MTT比色法检测结果显示:C组活力最高，A组活力低于与B组和C组;C组与A组比较有显著性差异(尸<0.05);C组OD值虽高于B组，但二者比较差异无显著性(P>0.05)。②GM一1对NSCs增殖的影响，MTT比色法检测结果显示:D组OD值高于B组，两者之间比较有显著性差异(P<0.05);E组OD值高于F组，两者比较有显著性差异(尸<0.05)，E组OD值与B组比较，无显著性差异 (P>0.OS)。③不同培养基、相同诱导分化条件下，分化结果显示:A组、C组分化后，NsE十率高于B组、D组和E组，GFAP+率低于B组、D组和E组;C组与A组间两项指标的比较无显著性差异(P>0.05)，C组与B组间两项指标的比较有显著性差异(尸<0.05):D组、E组与A组间的比较均有显著性差异(Po.05)。④同一培养基(B组)来源、不同诱导分化条件下分化结果显示:分化的4d和sd，bFGF组、LIF组NSE+率明显高于其余四组;LIF组与5%
Neural stem cells (NSCs), a type of stem cells, are capable of of self-renewal which may be maintained for life-long, proliferation and differentiation like all other stem cells. Accumulating evidence suggests that NSCs are characteristic to differentiate into three main kinds of central nervous system cells: neurons, astrocytes and oligodendrocytes. The discovery and intensive research of neural stem cells may bring new hope for curing the diseases of cental nerve system. But the proportions of NSCs vary at different developing phases and in different location of central nerve systems and are always relatively low. Since NSCs being discovered, neurologists all over the world have attempted to establish in vitro culture system to produce NSCs rapidly and to generate high rate of neurons through differentiating NSCs. Leukemia Inhibitory Factor( LIF), a 20 kD protein containing 180 ammo acid residues, is a multifunctional glycoprotein that induces macrophage differentiation and suppresses the proliferation of the murine Mlmyeloid cell line. LIF plays an important role, along with interleukin-6(IL-6) and granulocyte-colony stimulating factor(G-CSF), in the regulation of early hematopoietic stem cells. Gangliosides are one of the important component of cell membrane and CNS is rich of gangliosides. Both Gangliosides and polypeptide growth factors are trophic agents involved in almost all stages of neural cell generation, differentiation, survival, and pathology. In most cases their physiological roles remain unclear. Monosialotetrahexosylganglioside(GM-l), one of themain component of ganglioside in mammal capable of passing blood brain barrier and embedding in the damaged neurons membrane, has distinct functions in resisting on neuronal injury, promoting rehabilitation and growth of neural cells. In the recent years, most of the research regarding NSCs has focused on murine animals, while only a few on human embryonic NSCs. The influences of LIF on proliferation and differentiation of NSCs were carried out abroad , while there are few literatures available in our country . No report regarding the effects of GM-1 on NSCs is available in our country to date. In the present study, 10w human embryonic NSCs were isolated from ventricular zone/subventricular zone(VZ/SVZ) with mechanical method and incubated in serum-free medium with different additional components, the growth of each group was compared with other groups" and the proportions of neuron and glial cells of each group after differention under same or different condition were compared with other groups", thus the aptness of mechanical method for NSCs incubation and the influences of LIF and GM-1 on growth and differentiation of NSCs were discussed, moreover, Nestin and PCNA of the cells cultured and NSE and GFAP of the cells after differentiation were exmined according to immunocytochemistry method, in this way , the NSCs were identified.Methods: The tissues were obtained from VZ/SVZ zones(VZ/SVZ) of lOw human embryon and dismissed with a polish-Pasteur pipette. According to the difference of additional components in basic medium(DMEM/F-l 2+827), the cells were divided into five groups: A added with bFGF(20ng/ml), B with bFGF(20ng/ml)+EGF(20ng/ml), C with bFGF (20ng/ml) +EGF (20 ng/ml)+LIF(10ng/ml), D with bFGF(20ng/ml)+EGF(20ng/ml)+ GM-l(33.5ng/ml), E with GM-1 (33.5ng/ml), the cells of each group were put into two bottles and were cultured(5%CCO2, 37 ).(1)A few of cell suspension were got out of group A, B and C respectively, the cell concentrations were modulated to 1 105 /ml, then the cells of each group were plated in one 96 well culture plate and cultured in CO2 Gas Incubator, 12wells for each group and 0.1ml for each well . At 4d and 8d, the NSCs mobility of groups A, B and C were detected according to MTT method, then the influence of LIF on NSCs proliferation were learned.(2) A few of cell suspension were got out of group B, D, E and F (the basic medium-DMEM/F 12+827) respectively, the cell concentrations were modulated to 1 105 /
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