间充质干细胞（MSCs）神经向诱导实验条件优化及胞内钙离子浓度动态变化

发布时间：2018-10-12 08:26

【摘要】：目的：分离提取骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BMSCs)及脂肪间充质干细胞(Adipose-derived mesenchymal stem cells, AMSCs)比较二者生物学特性及体外增殖能力,并探讨三种不同成神经诱导方法对AMSCs向神经样细胞分化过程中的影响,提高AMSCs向神经样细胞分化诱导效率,从而为研究AMSCs向神经样细胞分化机制奠定基础。深入探讨成球诱导方法中细胞内钙离子浓度变化,有助于阐明AMSCs的成神经诱导分化机理,为AMSCs在临床再生医学上的应用提供理论依据。 方法：(1)采用密度梯度离心法分离BMSCs及酶消化法体外分离AMSCs。检测MSCs的表面抗原以及将MSCs进行体外成骨诱导,成脂诱导对MSCs的干细胞特性进行鉴定。(2)三种诱导方案诱导AMSCs向神经样细胞分化,包括化学诱导方法、鼠脑条件培养基诱导方法、成神经球分化诱导方法。观察诱导中的形态变化,免疫荧光检测神经细胞特异性标志物β-Ⅲ-Tubulin, NSE和Nissl的表达。确定最高效的诱导方法。(3)采取成神经球诱导方法对AMSCs进行诱导,利用Fluo3/AM对AMSCs及诱导过程中的类神经细胞进行染色,通过流式细胞仪对AMSCs群体细胞的钙离子浓度检测,并通过共聚焦显微镜确定单个细胞中钙离子浓度的变化,研究细胞内游离钙离子浓度在AMSCs向神经样细胞分化过程中的变化。 结果：(1)获得的BMSCs及AMSCs可稳定传至20代以上。表型鉴定结果为CD13、 CD90阳性,CD34、CD45阴性,AMSCs的CD106阴性,BMSCs CD106表达弱阳性。成骨细胞分化28d时,Von kossa阳性,向脂肪细胞分化14d时,油红O染色阳性。(2)用化学因子诱导AMSCs向神经样细胞分化,30d时出现成熟神经元标记NSE的表达,阳性值为29.5%,但细胞凋亡明显。鼠脑条件培养基诱导14d时,NSE略有表达,值为12.8%。采用成神经球诱导方法进行7d成神经球诱导,再经过7-9天的分化培养。在7d时有8.9%NSE表达。β-Ⅲ-Tubulin及Nissl在经三种诱导方法诱导成熟后,均为阳性表达。(3)成球法诱导AMSCs向神经样细胞分化过程中,取AMSCs及诱导不同时间点的成神经细胞从细胞整体及个体角度分别证实：在成球诱导法诱导下,通过7天成神经球后,在继续的分化过程1-6天时细胞内钙离子浓度先呈上升趋势,在分化7-9天时达到高峰,随后急剧下落,与细胞培养时细胞发生凋亡的时间一致。 结论：AMSCs较BMSCs具有较高的增殖能力,神经球分化方法更能够高效低损伤的诱导AMSCs分化为类神经细胞,细胞内游离钙离子在AMSCs向神经样细胞分化过程中会发生变化,对维持细胞分化,存活均起到了一定的作用。
[Abstract]:Objective: to isolate and extract bone marrow mesenchymal stem cells (Bone marrow-derived mesenchymal stem cells, BMSCs) and adipose mesenchymal stem cells (Adipose-derived mesenchymal stem cells, AMSCs) to compare their biological characteristics and proliferation ability in vitro. The effects of three kinds of neural induction methods on the differentiation of AMSCs into neuron-like cells were discussed, and the induction efficiency of AMSCs to neuron-like cells was improved, which laid a foundation for the study of the differentiation mechanism of AMSCs into neuron-like cells. It is helpful to elucidate the mechanism of neurogenesis and differentiation of AMSCs and provide theoretical basis for the application of AMSCs in clinical regenerative medicine. Methods: (1) BMSCs was separated by density gradient centrifugation and AMSCs. was isolated by enzyme digestion in vitro. The surface antigen of MSCs was detected and MSCs was induced by osteogenesis in vitro, and the characteristics of MSCs stem cells were identified by lipogenic induction. (2) three induction schemes induced AMSCs to differentiate into neuron-like cells, including chemical induction, brain conditioned medium induction. Methods of inducing neuronal differentiation. Morphological changes were observed and the expression of 尾-鈪，

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