大鼠骨髓间充质干细胞分离培养及分化为神经元样细胞的实验研究

发布时间：2018-05-05 18:35

本文选题：骨髓间充质干细胞 + 分离培养　；参考：《湖北中医学院》2009年硕士论文

【摘要】： 目的: 1.探讨大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的分离培养和体外扩增方法。 2.研究其在当归注射液或β-巯基乙醇(β-Mercaptoethanol,BME)诱导剂诱导下向神经元细胞分化的能力,以提供一种较好的诱导方案。 3.明确碱性成纤维生长因子(Basic fibroblast growth factor,bFGF)在诱导剂定向诱导过程中所起的作用,以优化诱导方案。 4.观察大鼠骨髓间充质干细胞的分离培养、体外扩增及定向诱导分化为神经元样细胞过程中的生物学特性,为今后临床研究提供良好的种子细胞来源。方法: 无菌条件下取大鼠骨髓,采用全骨髓贴壁培养筛选法分离出成年大鼠MSCs,进行体外培养、扩增,观察其生物学特性。取生长良好的第5代MSCs经碱性成纤维生长因子(bFGF)预诱导后用当归注射液或β-巯基乙醇(β-Mercaptoethanol,BME)诱导MSCs向神经元样细胞分化,并针对碱性成纤维生长因子(bFGF)在诱导过程所起作用设立三组实验对照组,包括对照组1:预诱导液中不含bFGF和诱导液中不含当归注射液或BME诱导剂;对照组2:预诱导液中含bFGF,而诱导液中不含当归注射液或BME诱导剂;对照组3:预诱导液中不含bFGF,而诱导液中含有当归注射液诱导剂。倒置显微镜下观察诱导过程中细胞形态所发生的变化,并通过细胞免疫化学法鉴定经各组诱导后所得细胞的神经干细胞标志物神经巢蛋白(neuroepithelial stem cell protein,Nestin)、神经元特异性烯醇化酶(Neuron-specific Enolase,NSE)、胶质纤维酸性蛋白(glialfibrillary acidic protein,GFAP)的表达。经镜下计数诱导分化后所得细胞的阳性表达率,用统计学q检验方法比较各组之间的差异。结果: 大鼠MSCs可用贴壁培养筛选法成功分离并在体外大量扩增。经当归注射液或BME诱导,MSCs可向神经元样细胞分化,均出现胞体和突起,细胞免疫化学染色神经元特异性烯醇化酶(NSE),巢蛋白(Nestin)阳性,神经胶质纤维酸性蛋白(GFAP)呈阴性。且经碱性成纤维生长因子(bFGF)预诱导的当归组较未经bFGF预诱导的对照组3诱导成神经元样细胞的阳性细胞表达率明显提高。结论: 贴壁分离培养法分离的MSCs能在体外培养条件下生长良好、可连续传代,体外培养扩增能力较强,以当归注射液或β-巯基乙醇作用于MSCs可成功诱导出神经元样细胞,MSCs能够成为理想的种子细胞来源。且碱性成纤维生长因子(bFGF)在诱导过程中起积极作用。
[Abstract]:Objective: 1. To investigate the isolation, culture and in vitro amplification of mesenchymal stem cells from rat bone marrow mesenchymal stem cells. 2. To study its ability to differentiate into neuron cells induced by Angelica sinensis injection or 尾 -Mercaptoethanolanol (BME) inducer, to provide a better induction scheme. 3. The role of basic fibroblast growth factor bFGFin in the induction of basic fibroblast growth factor (BFGF) was determined in order to optimize the induction scheme. 4. To observe the biological characteristics of rat bone marrow mesenchymal stem cells (BMSCs) in vitro amplification and differentiation into neuron-like cells and to provide a good source of seed cells for future clinical research. Methods: Adult rat MSCs were isolated from rat bone marrow in aseptic condition by whole bone marrow adherent culture screening method, and then cultured in vitro and amplified, and their biological characteristics were observed. The fifth passage of MSCs with good growth was preinduced by basic fibroblast growth factor (bFGF). MSCs was induced to differentiate into neuron-like cells by Angelica sinensis injection or 尾 -Mercaptoethanolol (尾 -Mercaptoethanol). According to the effect of basic fibroblast growth factor (bFGF) on the induction process, three experimental control groups were established, including control group 1: no bFGF in the preinduction solution and no angelica injection or BME inducer in the induced solution; In the control group, bFGF2 was found in the preinduction solution, but no angelica injection or BME inducer was found in the induced solution, while in the control group bFGF3 was not found in the preinducible solution, and the inducer in the induced solution was angelica sinensis injection. The changes of cell morphology during induction were observed under inverted microscope. The expression of Nestinan, Neuron-specific Enolase NSEE, glial fibrillary acidic protein (GFAPs) and neuron-specific enolase (Neuron-specific EnolaseNSEP) were detected by immunocytochemistry. The positive expression rate of the cells induced by differentiation was counted under microscope, and the difference between the groups was compared with the method of statistical Q test. Results: Rat MSCs was successfully isolated by adherent culture and expanded in vitro. BME could differentiate into neuron-like cells after Angelica sinensis injection or BME. The neuronal bodies and processes were found. The neuron-specific enolase (NSE) and nestin (nestin) were positive and glial fibrillary acidic protein (GFAP) was negative by immunocytochemistry. The expression rate of neuron-like cells in Angelica sinensis group preinduced by basic fibroblast growth factor (bFGF) was significantly higher than that in control group 3 without bFGF. Conclusion: The MSCs isolated by adherent culture method can grow well in vitro and can be subcultured continuously. MSCs treated with angelica sinensis injection or 尾 -mercaptoethanol could be used as an ideal seed cell source. Basic fibroblast growth factor (bFGF) plays an active role in the induction process.
【学位授予单位】：湖北中医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【引证文献】