NT3联合转化生长因子β诱导骨髓基质细胞分化为神经元样细胞的实验研究

发布时间：2018-10-08 21:14

【摘要】：前言骨髓基质细胞(Bone marrow stromal cells, BMSCs)是骨髓内的非造血干细胞,是造血干细胞分化增殖微环境的重要组成部分,具有自我更新和多向分化潜能,可以向多种来源于外胚层和中胚层的组织分化,形成骨、软骨、肌腱、脂肪和神经等组织细胞。在特定诱导条件下可转化为神经干细胞,并继续向神经元、星型胶质细胞和少突胶质细胞等细胞分化。另外有研究表明BMSCs能够通过血脑屏障,在脑中成活并迁移,促进多种神经营养因子的分泌,减少脑内细胞坏死和凋亡,促进脑创伤后神经功能的恢复,从而使得“自体骨髓源性神经干细胞”在中枢神经系统疾病的研究及应用成为可能,深入研究骨髓基质细胞的体外分化调控机制具有重要的临床意义。目前体外定向诱导骨髓基质细胞分化为神经元样细胞的实验条件尚未成熟,诱导分化率较低。因此,本实验从成年大鼠骨髓基质细胞的分离、纯化及扩增方案的实验参数优化出发,探讨NT3与TGF-β联合诱导骨髓基质细胞向神经细胞方向分化的可行性,为骨髓基质细胞的体外分化研究提供实验依据。材料与方法 1.1 主要试剂和仪器MSCGM培养基,TGF-β,NT3,bFGF(Hyclone公司),兔抗大鼠CD34,CD44单克隆抗体,Nestin、NSE、β-catenin、GFAP(Sigma公司),胰蛋白酶,胎牛血清,辣根酶标记链亲和素,DAB显色液,封闭用山羊血清,二抗试剂盒(华美公司)。CO_2恒温培养箱,生物净化工作台,离心机,干燥箱,组化常用器皿,Olympus CK40倒置显微镜。 1.2 骨髓基质细胞的分离、培养和鉴定在无菌条件下取1个月龄Wistar大鼠股骨和胫骨的骨髓细胞做原代培养。根据骨髓基质细胞与其他细胞的密度不同而采用Percoll分离液将其分离,根据细胞贴壁不同,筛选易
[Abstract]:Bone marrow stromal cells (Bone marrow stromal cells, BMSCs) are non-hematopoietic stem cells in bone marrow and an important part of microenvironment for differentiation and proliferation of hematopoietic stem cells. It has the potential of self-renewal and multi-differentiation, and can differentiate into various tissues from ectoderm and mesoderm, forming bone, cartilage, tendon, fat and nerve tissue cells. Under certain induction conditions, neural stem cells can be transformed into neural stem cells and continue to differentiate into neurons, astrocytes and oligodendrocytes. In addition, some studies have shown that BMSCs can survive and migrate in the brain through the blood-brain barrier, promote the secretion of various neurotrophic factors, reduce the necrosis and apoptosis of brain cells, and promote the recovery of neural function after brain trauma. This makes it possible to study and apply autologous bone marrow-derived neural stem cells in central nervous system diseases, and it is of great clinical significance to study the mechanism of differentiation and regulation of bone marrow stromal cells in vitro. At present, the condition of inducing bone marrow stromal cells to differentiate into neuron-like cells in vitro is not mature, and the rate of differentiation is low. Therefore, the feasibility of NT3 combined with TGF- 尾 inducing the differentiation of bone marrow stromal cells into neural cells was discussed from the experimental parameters of isolation, purification and amplification of bone marrow stromal cells from adult rats. To provide experimental basis for in vitro differentiation of bone marrow stromal cells. Materials and methods 1. 1 the main reagent and instrument MSCGM medium: TGF- 尾 nt3bFGF (Hyclone), rabbit anti-rat CD34,CD44 monoclonal antibody NSE, 尾 -cateninine GFAP (Sigma), trypsin, fetal bovine serum. Horseradish enzyme labeled chain avidin dab chromogenic solution, goat serum, second antibody kit (Huamei company). COS2 constant temperature incubator, biological purification table, centrifuge, drier, histochemical utensils, Olympus CK40 inverted microscope. 1.2 Bone marrow stromal cells were isolated, cultured and identified under aseptic conditions. Bone marrow cells from femur and tibia of 1 month old Wistar rats were taken for primary culture. According to the density of bone marrow stromal cells and other cells, the Percoll isolate was used to isolate them. According to the different adherent cells, it was easy to screen.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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