论文标题:大鼠骨髓间充质干细胞生物学特性、体外神经元方向分化及静脉移植治疗大鼠脑损伤的实验研究
Research of the Biologic Characteristics, Neural Differention of Rat Bone Marrow Mesenchymal Stem Cells(MSCs) in vitro and Experimental Investigation of Curability of Rat with Brain Injury by MSCs Transplanted Via Vein
论文作者 王宁
论文导师 杨树源,论文学位 博士,论文专业 神经外科学
论文单位 天津医科大学,点击次数 64,论文页数 125页File Size8089k
2003-05-01论文网 http://www.lw23.com/lunwen_90069417/ 大鼠,颈动脉,血管成形术,导丝,球囊,再狭窄模型,血管,外膜,成纤维细胞,肌成纤维细胞,迁移,血管中膜,血管内膜,新生内膜,血管造影,磁共振成像,血小板衍化生长因子,血小板衍化生长因子受体,新生内膜,寡核苷酸,反义,表达,表型
rat,carotid artery,angioplasty,guidewire,balloon,restenosis,model,adventitia,flbroblast,myofibroblast,migration,media,intima,neointima,digital substrct angiography,magnetic resonance imaging,platelet-derived growth factor,platelet-derived growth factor receptor,oligodeoxynucleotide,antisense,phenotype,expression
第一部分 rMSCs生物学特性的研究 骨髓中含有两大类细胞群，一个是能产生红细胞、血小板、单核细胞、粒细胞的造血干细胞(hematopoietic stem cells，HSCs)，它为循环血液提供前体细胞(或称为祖细胞)，这是人所共知的。另一个就是同样符合干细胞定义的非造血性干细胞。这种干细胞样的细胞被称为骨髓基质细胞(bone marrow stromal cells，BMSCs)或间充质干细胞(mesenchymal stem cells，MSCs)。20世纪70年代中期，Friedenstein的先驱性研究揭示了骨髓中存在非造血干细胞这一事实，其后的二十年来，虽然对MSCs多向分化能力的特性了解不断加深，但仍然有不少疑问有待进一步研究。我们对MSCs的分离，培养条件下的生长特性及表面抗原标志进行了观察研究。 利用MSCs具有贴壁生长的特性，分离MSCs。光学及电子显微镜下观察细胞形态。MTT法，测定MSCs生长曲线。以16／cm~2和3／cm~2两种不同的细胞接种密度，并添加EGF，bFGF，EGF+bFGF，生长18天后，记数生成的细胞克隆及细胞数。流式 天津医科大学博士学位论文细胞仪检测MSCs免疫细胞表型。 全骨髓细胞接种后，通过更换培养液，弃去悬浮生长的造血细胞，ro一14天后，MSCs形态上趋于一致，光镜下呈梭形或纺睡形的成纤维样细胞。以3个细胞/c扩、16个细胞/c mZ不同的细胞密度接种于75c扩底面积培养瓶，培养18天后，3个细胞/c扩和16个细脚cmz组，在Q一MEM中，按照接种单位细胞产量(/ Icell)生成的细胞数分别为:56.89士8.55，22.5士2.64; 克隆数(八OOeells)分别为1 .69士0.37，0.60士0.11。3个细胞/c扩，明显多于16个细脚cmZ组，统计学分析表明差异有显著性，表明接种密度对MSCs的影响。EGF、EGF+bFGF对细胞增殖有显著的促进作用。流式细胞仪免疫细胞表型检测表明MSCs 83.1%表达CD90，80%表达CD71;只有3.53%表达CD45;3.51%表达CD 14;3.71%表达 CD34;4.67%表达HLA一DR。 MSCs可以通过其贴壁的特性加以分离，MSCs能较长时间传代培养并且其形态不发生明显改变，从形态学上尚不能鉴别MSCs，免疫细胞表型测定有助于识别MSCs。低密度接种培养可获得较多的细胞量，EGF能刺激MSCs快速增殖。第二部分rMSCs体外诱导分化为神经元样细胞骨髓中的HSCs能持续补充循环血细胞;MSCs通常只产生间 天津医科大学博士学位论文叶源的细胞。发育过程实际上是细胞命运的进行性限制过程:早期胚胎细胞是全能的，而出生后动物的成体干细胞，即组织特异性干细胞，据认为只能分化为它们所存在组织中的细胞类型。MSCs是经典的内胚层细胞，它代表了一个独特的细胞群，尽管不是全能的，但它们可以自我更新，并分化为不止一种特异的细胞类型。在体内、体外可以分化为传统上认为属于外胚层的细胞如神经元，胶质细胞。这些研究动摇了发育生物学的经典理论，这意味着这些细胞可以再程序化而转分化(transdifferentiated)为其它胚层的细胞。我们研究MSCs在体外培养中，在多种诱导因子的作用下，MSCs向神经细胞分化的能力。 对MSCs3~4代培养细胞分别以3种神经诱导培养液诱导，光学显微镜下观察细胞形态变化，诱导培养6天后，细胞进行神经元特异抗体免疫组化染色鉴定，了解细胞分化的情况，记数MSCs分化为神经元的比率。 细胞在诱导后30科O分钟即开始出现变化，细胞体收缩，细胞从梭形变为圆形，细胞折光性增强，并有突起形成，细胞有单极，双极及多极神经元样细胞出现。随时间增加，神经元样细胞的突起可连成网状。对用诱导培养液3诱导6天的细胞进行免疫组化染色，神经元样细胞表达NSE，NeuN，及MAP一2，表明其有神经元蛋白表达，NSE、MAP一2，NeuN的表达率分别为59.83%， 天津医科大学博士学位论文45.17%和50.83%。 MSCs在神经诱导培养液中能很快分化成神经元样的细胞，免疫组化染色这些细胞表达神经元特异性蛋白，这种由细胞外信号触发的分化是快速的，说明MSCs本身具有神经元的基因表达序列，MSCs是多能的，具有向神经外胚层分化的能力。 第三部分rMSCs静脉移植治疗大鼠脑损伤 进行神经移植的主要障碍在于有限的供体组织。异体胚胎，胎)匕神经干细胞是一个理想的移植组织，但采集细胞及移植时间窗较短，伦理及法律原因也阻碍了胚胎组织用来进行广泛的神经移植。MSCs是一个潜在的能应用于移植，并能诱导内源性干细胞增殖的细胞。使用自体的MSCs进行移植，避免了外源物质的进入，也避免了伦理学，法律方面的障碍，并可显著减少免疫抑制剂的应用。我们将大鼠MSCs经尾静脉注射移植入脑创伤大鼠，了解骨髓源性细胞在脑内的迁移情况，以及对大鼠神经运动功能的影响。 以自由落体打击法制作诚star大鼠脑损伤模型，分4组，将BrdU标记MSCs，以3 x 106细胞/ml，在大鼠伤后l天(第1组，n=6)，3天(第2组，n=Io)，7天(第3组，n=7)及未损伤大鼠 (第4组，对照组，n=3)，l血经尾静脉注射入大?
Part one. Research of the biologic characteristics of rat bone marrow mesenchymal stem ceUs (MSCs) in vitroBone marrow contains two prototypical stem cell populations. It"s well known that one of them is hematopoietic stem cells, which provide a continuous source of progenitors for red cells, platelets, monocytes, and granulocytes. Another is the cells that meet the criteria for stem cells of non-hematopoietic precursors. These stem-like cells currently referred to either as bone marrow stromal cells (BMSCs) or as mesenchymal stem cells (MSCs). In the mid-1970s the pioneering work of Friedenstain discovered the fact that bone marrow contains the non-HSCs. Although the potential of multi-differentiation properties of MSCs have been recognized for two decades, there are still many questions that need to be studied further. In this paper thebiological characteristics of MSCs, such as the isolations, culture conditions, growth features and cells surface markers have been observed and studiedThe MSCs from Wistar rat were isolated by its characteristics of growing adherence to the plastic surface with tissue culture. The growth line was measured by MTT method. The growth features of MSCs were observed when cells were plated at two densities of 3 cells/cm2, 16 cells/cm2 with adding in EOF, bFGF and EGF+bFGF culture conditions. After 18 growth days the number of cells, CFU-Fs had been measured. The immunophenotype of MSCs were detected byFCM.Rat bone marrow, taken from femurs and tibias, were plated in a-MEM medium. Through the change of medium, HSCs were removed. The morphology of MSCs tended homogenous after 10~14days. Cells have fibroblastic-like morphology. MSCs were plated in a 75cm2 clulture flask with densities of 3 cells/cm2 and 16 cells/cm2, after 18 days the total yield per cell plated in a-MEM medium was approximately 56.89± 8.55 and 22.5 ± 2.64, respectively. The yield of CFU-F per 100 cells plated was 1.69±0.37 and 0.60±0.11, respectively. The growth factors, suchas EOF, bFGF stimulated the proliferation of MSCs. FCM showed that 83.1% of MSCs isolated by our method expressed CD90 and 80% of cells expressed CD71.From our experiment it can be concluded that MSCs can be isolated by its characteristic of adherence to the wall. It can be cultured in a long-term without obviously changing its morphology. There is still an obstacle to distinguish the cells from its morphology. Immunological cell phenotype assay is helpful to recognize MSCs. When MSCs were plated at different density, the yield of cells and CUF-F are different. Culturing in low density can produce more cells. The stimulating growth factors can also increase the cells production.Part two. Study on the neural differentiation of rat bone marrow mesenchymal stem cells (MSCs) in vitroHSCs in bone marrow continuously repopulate the blood cells of the circulation. While MSCs, the mesodermal elements normally give rise only to mesenchymal derivatives. Developmental procedure of cells is actually a progressive restriction procedure to cells fate. Early embryonic cells are totipotent. Postnatal stem cells are thought to differentiate only into cells of their resident tissues. MSCs, classicalmesodermal derivatives, represent a rare population in postnatal animals. Although not totipotent, they are capable of self-renewal and differentiate into more than one specialized cell type. It can differentiate into neurons and glia, traditional ectodermal cell in vivo and in vitro. These studies have rocked the developmental biologic theories that have been accepted for hundreds of years. It means that cells can reprogram or trans-differentiate from one germ layer to another. The capability of MSCs differentiate into neural cells under the induction of some factors in vitro has been studied in this paper.The brief experimental process is as follow: MSCs(P3~P4) were maintained in sub-confluent cultures in NIM with inducing the neuronal phenotype for 6 days, during this time the morphological
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