骨髓与脂肪间质干细胞神经向分化能力比较研究

发布时间：2018-04-23 01:01

  本文选题：骨髓间充质干细胞(bone + marrow　； 参考：《大连医科大学》2010年硕士论文

【摘要】： 目的：神经系统疾病如脊髓损伤、退行性病、多发硬化、运动神经元病等,主要原因是神经细胞的坏死、功能的缺失。干细胞(stem cells)是一类具有无限自我更新和多向分化潜能的细胞,在一定条件下,可分化为神经元样细胞和神经胶质样细胞,有望作为神经系统疾病细胞移植的种子细胞,但是干细胞种类繁多,据其发生学来源的不同可分为胚胎干细胞(embryonic stem cells, ESCs)和成体干细胞(adult stem cells, ASCs),其中ASCs是指存在于已分化组织的未分化细胞。ASCs较ESCs的优点是取材相对方便,不存在伦理学问题。目前研究较多的成体干细胞主要是骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs),它取材方便,易于分离纯化,神经向分化能力的研究也比较多,已应用于前期临床研究试验,而脂肪间充质干细胞(Adipose-derived Mesenchymal Stem Cells,ADSCs)作为近年来新发现的种子细胞,神经向分化能力的研究还相对较少,但它具有BMSCs所不具备的优点,如：获取更为方便,对人体创伤小,很少受血液及免疫系统的影响等。本实验旨在比较人类BMSCs与ADSCs的神经向分化能力,为干细胞移植治疗神经系统疾病筛选理想种子细胞。 方法：1)采用Percoll分离法在体外培养及扩增BMSCs,利用胰酶消化的方法获取ADSCs。通过表面抗原检测的方法进行鉴定。2)采用多种生长因子联合诱导方案诱导ADSCs、BMSCs神经向分化,即先用浓度为10 ng/ml的bFGF预诱导24 h,再加入20 ng/ml的bFGF、20 ng/ml的EGF、20 ng/ml的BDNF,分别于24 h、3 d、7 d、10 d、14 d时进行免疫荧光检测Nestin,β-Ⅲ-Tubulin、NSE的表达,尼氏荧光染色检测尼氏小体表达,观察细胞形态变化。3)统计阳性细胞的比率,卡方检验分析统计结果。 结果：1.采用percoll密度梯度离心法成功分离出人骨髓间充质干细胞,细胞表型鉴定为CD90阳性,CD106阳性,CD34阴性。2.通过对比ADSCs和BMSCs的生长曲线发现,脂肪间充质干细胞较骨髓间充质干细胞具有更强的增殖能力。3.利用生长因子联合诱导的方式诱导BMSCs和ADSCs神经向分化,诱导后,BMSCs和ADSCs细胞形态都发生了改变,胞体变圆并伸出细长突起,对于分化细胞的神经细胞特异标记的鉴定也具有相同的趋势：诱导中期Nestin、β-Ⅲ-tublin显著增多,晚期稍降,而NSE和Nissl小体诱导晚期才出现具有相似的阳性率。 结论：1.ADSCs具有较BMSCs更为活跃的生长、增殖能力；2.ADSCs具有与BMSCs类似的神经向分化能力。
[Abstract]:Objective: neurologic diseases such as spinal cord injury, degenerative disease, multiple sclerosis, motor neuron disease, etc. Stem cells are a kind of cells with infinite self-renewal and multi-differentiation potential. Under certain conditions, stem cells can be differentiated into neuron-like cells and glial cells, which may be used as seed cells for transplantation of cells in nervous system diseases. However, stem cells can be divided into embryonic stem cells (ESCs) and adult stem cells (adult stem cells) according to the different origin of stem cells. ASCs refers to undifferentiated cells that exist in differentiated tissues. ASCs are more convenient than ESCs. There is no ethical problem. At present, the main adult stem cells studied are bone marrow mesenchymal stem cells (BMSCs), bone marrow mesenchymal stem cells (BMSCs). As a newly discovered seed cell in recent years, adipose mesenchymal stem cells (Adipose-derived Mesenchymal Stem cells ADSCs) have relatively little research on the ability of neural differentiation, but it has the advantages that BMSCs does not. For example, it is more convenient to obtain and less invasive to human body. Rarely affected by the blood and immune system, etc. The purpose of this study was to compare the neural differentiation ability of human BMSCs and ADSCs and to screen ideal seed cells for stem cell transplantation in the treatment of nervous system diseases. Methods BMSCs were cultured and amplified by Percoll in vitro, and ADSCs were obtained by trypsin digestion. The neural differentiation of ADSCsBMSCs was induced by a combination of multiple growth factors and the detection of surface antigen. The expression of Nestin, 尾-鈪，

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