骨髓间充质干细胞神经分化的体内外研究

目的研究骨髓间充质干细胞在体内外向神经细胞分化的潜能。方法(1)分离培养：从水囊引产胎儿的四肢骨中分离培养骨髓基质细胞，再通过贴壁方法将间充质干细胞从骨髓来源的单个核细胞中分离出，利用流式细胞仪进行间充质干细胞免疫表型测定。(2)体外分化观察：将间充质干细胞在二甲基亚砜(DMSO)和丁羟茴醚(BHA)中作用5h后，行神经元特异性烯醇化酶、神经微丝等免疫组化染色。(3)体内分化观察：将用5一溴脱氧尿核苷(BrDU)标记的间充质干细胞通过立体定向的方法移植于顶叶皮质区，然后再行烯醇化酶、神经微丝及胶质纤维酸性蛋白免疫组化染色。结果(1)生物学特性：间充质干细胞贴壁生长，细胞表现为梭形细胞形态，骨髓来源的间充质干细胞CD34、HLA—DR的表达为阴性，而CD44、CD29、CD13的表达为阳性。(2)体外分化：间充质干细胞在体外被DMSO／BHA处理后，胞浆向细胞核方向收缩，形成一个收缩的胞体，外周是细胞膜的突起，呈现出典型的神经元表型，并且免疫组化染色呈现神经元特异性烯醇化酶及神经微丝染色阳性。(3)体内分化：间充质干细胞被立体定向移植后，在移植部位可见BrDU染色阳性的细胞团，同时可见BrDU阳性细胞迁移到周围宿主的皮质中。免疫组化染色显示，在移植物中及邻近宿主皮质中的移植细胞呈神经元标记烯醇化酶染色阳性，星形细胞标记胶质纤维酸性蛋白染色阳性。结论问充质干细胞有能力分化为非间充质细胞，特别是神经元。

Study on differentiation of bone marrow mesenchymal stem cells in vitro and in vivo

Objective To study the potential of bone marrow mesenchymal stem cells (MSCs) differentiating into nerve cells in vitro and in vivo. Methods (1) Bone marrow stromal cells were isolated from bone marrow of aborted fetus extremities and cultured in vitro, which MSCs were separated by adhesion to wall of culturing discs, and the immune phenotype of MSCs were identified by flow cytometer. (2) The MSCs were stained by neuron specific enolase (NSE) and neurofilament (NF) immunohistochemical staining after treated with dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) for 5h and then observed in vitro. (3) The 5-bromodeoxyuridine (BrDU) labled MSCs were stereotaxic transplanted into the parietal cortex of Wistar rats, then stained by enolase, NF and glial fibrillary acidic protein immunohistochemical staining. Results (1) Cytological characteristics cultured MSCs were adhesive to the wall of culturing discs and appeared as shuttle-like shape, the bonemarrow origin MSCs were demonstrated positive CD44, CD29 and CD13 expression and negative CD34 and HLA-DR expression. (2) Differentiation in vitro after treated with DMSO/BHA in vitro, the cytoplasm of MSCs were shrunken toward cell nucleus. The cells surrounded by cell membrane processes were showed as typical phenotype of neurons and demonstrated positive immunoreactivity of NSE and NF in immunohistochemical staining. (3) BrDU staining positive cells were detected at graft site, which could migrate into host cortex nearby. Immunohistochemical staining showed that the transplanted cells in graft and adjacent host cortex were NSE positive or GFAP positive. Conclusion The MSCs are capable to differentiate into nonmesenchymal cells especially neurons.