Bone marrow mesenchymal stem cells differentiate into neuron-like cells induced by combination of two cytokines

doi:10.3969/j.issn.2095-4344.2014-06-002

Abstract

Abstract:

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) can be induced to differentiate into neuron-like cells directionally. Accordingly, BMSCs can be used as seed cells theoretically in constructing tissue-engineered peripheral nerves.

OBJECTIVE: Using combination of two cytokines to induce BMSCs differentiating into neuron-like cells directionally, and further to discusse its application in peripheral nerve injury.

METHODS: BMSCs were isolated and purificated from the bone marrow of Wistar rats by using the differential adherence method. Basic fibroblast growth factor and epidermal growth factor were used to induce the BMSCs differentiating into neuron-like cells. The morphological change was observed and the neuronal specific markers were detected by immunohistochemistry technique. The morphological and immunohistological changes were also studied after the induce agent were removed.

RESULTS AND CONCLUSION: With presence of morphological and immunohistochemical features of nerve cells induced by neurotrophic factors, BMSCs exhibited two or more processes that were interconnected as a meshwork; cell nucleus and nucleus could be observed with strong light refraction of cytoplasm. After immunohistochemical staining, neuroln specific enolase, neurofilament protein and synaptophysin protein positive cells were detected. A great amount of cells reversed to their original fibroblast-like morphology, and the expression of the three above-mentioned proteins decreased as the induce agent withdrawn. Our study showed that BMSCs can be induced to differentiate into neuron-like cells, but the transdifferentiation is a short-time reversible phenomenon.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: neurturin, mesenchymal stem cells, mesenchymal stem cell transplantation, cell differentiation

CLC Number:

R394.2

Huang Jian-feng, Huang Ji-feng, Zhang Wei-cai. Bone marrow mesenchymal stem cells differentiate into neuron-like cells induced by combination of two cytokines[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(6): 829-834.

Figures/Tables 3

2.3 免疫组化结果进一步确定骨髓间充质干细胞的神经分化为进一步确定骨髓间充质干细胞是否分化为神经细胞，分别于各观察时相点采用神经元特异标志物神经元特异性烯醇化酶、神经丝蛋白，突触囊泡膜特异性蛋白质突触素进行鉴定[35-37]。免疫组化结果显示，碱性成纤维生长因子及表皮生长因子联合诱导5 d后，大部分有核细胞胞质表达神经元特异性烯醇化酶，染色呈红色(图2A)，部分细胞突起延长相互交织成网状。神经丝蛋白是一种神经元特异性的中间纤维，是神经元胞质的主要结构之一[36]，其阳性表达部位与神经元特异性烯醇化酶类似，染色呈绿色，主要存在于细胞胞体与突起部位(图2D)。突触素蛋白是存在于突触囊泡膜上的一种糖蛋白质，参与突触发生[37]，其表达主要集中在胞质，染色呈绿色(图2G)。联合诱导8 d后，细胞呈典型的神经细胞形态，神经元特异性烯醇化酶的表达有所下降(图2B)，神经丝蛋白和突触素的表达上升，主要集中在胞质，甚至是部分突触(图2E，H)。撤除诱导条件3 d后，大部分细胞逐渐恢复原来的成纤维细胞形态，上述3种蛋白的表达量均明显下降(图2C，F，I)。对照组细胞未见3中蛋白阳性表达(图2J，K，L)。各个观察时相点3种蛋白的阳性率：诱导5 d后，神经元特异性烯醇化酶、神经丝蛋白、突触素阳性率表达分别为(70.3±4.2)%，(25.7±1.6)%，(3.6±0.5)%；诱导8 d后，神经元特异性烯醇化酶、神经丝蛋白、突触素阳性率表达分别为(54.1±3.8)%，(47.1±2.3)%，(32.0±2.1)%，与诱导5 d后比较差异有显著性意义(P < 0.01)；撤除诱导条件3 d后，神经元特异性烯醇化酶、神经丝蛋白、突触素阳性率表达分别为(21.6±2.3)%，(13.8±1.7)%，(9.4±1.1)%，与诱导5 d、诱导8 d后比较差异均有显著性意义(P < 0.01，图3)。"

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