全骨髓法分离培养骨髓间充质干细胞及向神经样细胞分化的研究

目的目前尚缺乏标准的骨髓间充质干细胞（BMSCs）的分离方法,实验通过全骨髓培养法分离纯化骨髓间充质干细胞,并诱导分化为神经样细胞,以寻找一种简便、实用的分离方法。方法取大鼠骨髓,全骨髓培养法结合差速贴壁法分离纯化大鼠骨髓间充质干细胞,传代扩增。倒置相差显微镜下观察骨髓间充质干细胞形态及生物学特点,免疫组化染色鉴定细胞。用MTT法间接测定不同代数细胞活力。诱导分化,先用碱性成纤维细胞生长因子（bFGF）预诱导24h,然后加入丁羟基茴香醚诱导（BHA）和二甲基亚砜（DMSO）诱导。观察诱导过程中细胞的形态变化,并用免疫组化染色法鉴定细胞。结果全骨髓培养法成功分离培养BMSCs,免疫荧光染色示CD90、CD71、CD106表达阳性,CD45阴性。MTT法检测细胞活力示2、4、6代细胞于酶标仪490nm波长处吸光度值均高于第8、10代细胞。细胞经诱导后,细胞呈神经样细胞形态,并且神经元特异性表面标志β微管蛋白（β-Ⅲ-Tubulin）、巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）表达阳性。结论采用全骨髓培养法可获得较纯化的骨髓间充质干细胞,其在适宜的诱导分化条件下可诱导分化为神经样细胞。

The sdudy of differentiation of bone marrow mesenchymal stem cells into neural stem cells by whole bone marrow adherence method

Objective At present there have not been standard separation method of mesenchymal stem cells （BMSCs）, the experiment further validated that BMSCs could be isolated with the method of whole bone marrow adherence method, and then differentiated into neuron-like cells, to find a simple, practical method of separation. Methods BMSCs were isolated and purified by whole bone marrow adherence method,the morphology of BMSCs was observed and the expression of cell surface antigens of BMSCs were analyzed with immunohistochemical method. Cell activity was indirectly detected by MTT method. The BMSCs were induced into neuron-like cells with basic fibroblast growth factor （bFGF） for 24h,and then by hydroxy anisol （BHA） and dimethyl suphoxide （DMSO）, the process of differentiation was observed. Specific markers of induced cells were de- tected by immunofluorescence. Results BMSCs were isolated and purified successfully and effectively,the results of the iden- tification were positive of CD90, CD106, CD71, while CD45 was negative. MTT assay showed that there was no significant dif- ference in absorbance in 2,4,6 passages of cells in 490nm wavelength by enzyme-labeled instrument, but the absorbance was higher than that of 8 or 10 passage of cells. After neurogenic induced by DMSO and BHA,the morphology of BMSCs had be- come neuron-like cells, immunohistochemically analysis showed that nerve cell surface marker were positive of Nestin, β-Ⅲ- Tubulin and NSE. Conclusion BMSCs can be successfully cultivated by whole bone marrow adherence method. Under proper condition, BMSCs may differentiate into neuron-like cells.