骨髓间充质干细胞的分离培养及脑脊液对骨髓间充质干细胞的影响
发布时间：2018-07-17 08:09
【摘要】： 目的:探讨体外分离、纯化大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells BMSCs)的方法及骨髓间充质干细胞向神经元样细胞的分化潜能,并分析其部分表型特点。动态观察体外脑脊液对骨髓间充质干细胞的影响,为骨髓间充质干细胞的移植提供初步的实验依据。 方法: 1.选取清洁级SD大鼠,体重150g～200g。用密度梯度离心结合贴壁培养法及全骨髓培养法分离纯化大鼠骨髓间充质干细胞,并传代扩增。进行形态学观察,免疫组化法分析测定细胞表面抗原的表达。2.采用抗氧化剂诱导方案诱导骨髓间充质干细胞向神经元样细胞分化,观察诱导过程中细胞的形态变化,并用免疫荧光法检测神经样细胞的特异性标志物表达,即免疫组化法检测诱导后神经元样细胞的特异性烯醇化酶、微管相关蛋白和巢蛋白的表达。3.选择生长良好的第3、4代细胞接种于脑脊液中,观察细胞生长状况,并通过细胞免疫化学染色法鉴定脑脊液对细胞表型的影响。制定细胞生长曲线。 结果: 1. BMSCs属骨髓中单个核细胞,运用密度梯度离心结合贴壁培养法及全骨髓培养法能成功有效的分离纯化大鼠BMSCs,并可稳定传至20代以上。表型鉴定结果为CD90、CD106、CD71、CD29阳性,CD45阴性。2.采用bFGF/BHA诱导BMSCs向神经样细胞分化,诱导过程中细胞形态学发生了改变,bFGF诱导24h后细胞由扁平和长梭状变为多边形及不规则形。加入DMSO和BHA5h后,胞体变圆,折光率增加,并伸出有2到3或更多突起。诱导24h后细胞形态更像神经元,并交织呈网状。诱导后神经细胞的标志物Nestin,β-Ⅲ-Tubulin和NSE表达阳性,并且BMSCs的表面标记CD106表达阳性。3.骨髓间充干质细胞在脑脊液培养基中仍可继续生长、增殖,提示这种细胞对生长环境有高度的适应性,并不影响其特性和表型。 结论:本实验建立了一种体外分离纯化、培养扩增大鼠骨髓BMSCs的方法,用Percoll淋巴细胞分离液分离培养BMSCs可以快速扩增和纯化,并能稳定传代培养。采用bFGF/BHA诱导方案,能成功诱导骨髓间充质干细胞分化为神经元样细胞。骨髓间充质干细胞对脑脊液有高度的适应性,可作为一种新的培养方法。
[Abstract]:Aim: to investigate the method of isolation and purification of rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells BMSCs) in vitro and the differentiation potential of bone marrow mesenchymal stem cells into neuron-like cells. To observe the effect of cerebrospinal fluid (CSF) on bone marrow mesenchymal stem cells (BMSCs) in vitro and to provide a preliminary experimental basis for the transplantation of BMSCs. Methods: 1. SD rats of clean grade were selected, weighing 150 g or 200 g. Rat bone marrow mesenchymal stem cells were purified by density gradient centrifugation combined with adherent culture and whole bone marrow culture. Morphological observation and immunohistochemical analysis were used to detect the expression of cell surface antigen. 2. 2. Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into neuron-like cells by antioxidant induction. The morphological changes of the cells were observed and the expression of specific markers of neuron-like cells was detected by immunofluorescence. The expression of specific enolase, microtubule-associated protein and nestin in neuron-like cells was detected by immunohistochemistry. The third generation of well growing cells were inoculated into cerebrospinal fluid (CSF) to observe the growth of the cells and to identify the effect of CSF on the phenotype of the cells by immunocytochemical staining. Draw up cell growth curve. Results: 1. BMSCs are mononuclear cells from bone marrow. Using density gradient centrifugation combined with adherent culture method and whole bone marrow culture method, BMSCs can be separated and purified effectively, and can be transferred to more than 20 generations. The results of phenotypic identification were CD90, CD106, CD71, CD29 positive and CD45 negative. 2. BMSCs were induced to differentiate into neural like cells by bFGF- / BHA. The morphology of BMSCs changed from flat and long spindle to polygonal and irregular shape after 24 hours of induction by bFGF. After the addition of DMSO and BHA for 5 h, the cell body became round, the refractive index increased, and there were 2 to 3 or more protrusions. After 24 hours of induction, the cells were more neuronal in shape and intertwined in a reticular form. After induction, Nestin, 尾-鈪，
本文编号：2129738
[Abstract]:Aim: to investigate the method of isolation and purification of rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells BMSCs) in vitro and the differentiation potential of bone marrow mesenchymal stem cells into neuron-like cells. To observe the effect of cerebrospinal fluid (CSF) on bone marrow mesenchymal stem cells (BMSCs) in vitro and to provide a preliminary experimental basis for the transplantation of BMSCs. Methods: 1. SD rats of clean grade were selected, weighing 150 g or 200 g. Rat bone marrow mesenchymal stem cells were purified by density gradient centrifugation combined with adherent culture and whole bone marrow culture. Morphological observation and immunohistochemical analysis were used to detect the expression of cell surface antigen. 2. 2. Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into neuron-like cells by antioxidant induction. The morphological changes of the cells were observed and the expression of specific markers of neuron-like cells was detected by immunofluorescence. The expression of specific enolase, microtubule-associated protein and nestin in neuron-like cells was detected by immunohistochemistry. The third generation of well growing cells were inoculated into cerebrospinal fluid (CSF) to observe the growth of the cells and to identify the effect of CSF on the phenotype of the cells by immunocytochemical staining. Draw up cell growth curve. Results: 1. BMSCs are mononuclear cells from bone marrow. Using density gradient centrifugation combined with adherent culture method and whole bone marrow culture method, BMSCs can be separated and purified effectively, and can be transferred to more than 20 generations. The results of phenotypic identification were CD90, CD106, CD71, CD29 positive and CD45 negative. 2. BMSCs were induced to differentiate into neural like cells by bFGF- / BHA. The morphology of BMSCs changed from flat and long spindle to polygonal and irregular shape after 24 hours of induction by bFGF. After the addition of DMSO and BHA for 5 h, the cell body became round, the refractive index increased, and there were 2 to 3 or more protrusions. After 24 hours of induction, the cells were more neuronal in shape and intertwined in a reticular form. After induction, Nestin, 尾-鈪，
本文编号：2129738
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