课题背景：课题为2006年泸州医学院青年自然科学基金项目，由于骨髓基质细胞取材方便，对机体无害，取自自体，避免免疫排斥反应，因此课题采用骨髓基质细胞体外诱导神经干细胞，为在体修复神经系统损伤奠定实验基础。目前课题项目已完成骨髓基质细胞体外诱导分化为神经干细胞，并将诱导的细胞移植入实验性变态反应性脑脊髓炎大鼠模型。

应用要点：①对于神经系统疾病的神经干细胞移植治疗，需要大量纯化的细胞。课题将骨髓基质细胞在类似诱导神经干细胞的条件下体外诱导分化，以期获得满足条件的神经干细胞，为神经系统疾病的治疗提供种子细胞。②实验所用诱导方法简单易行，在体外即可诱导获得大量纯化神经干细胞供细胞移植。③体外诱导的神经干细胞又可在适宜诱导条件下向某一类神经细胞或胶质细胞的分化。

偏倚与不足：实验仅就体外诱导大鼠骨髓基质细胞向神经干细胞方向分化进行了探讨，还需进一步分析诱导分化所得神经干细胞体内移植治疗某一具体神经系统疾病后，细胞在宿主体内的存活、迁移及分化情况，以及如何促进受损组织重塑和神经功能恢复，从而为多种神经系统疾病的神经干细胞移植治疗提供基础研究资料。

摘要
目的：已证实骨髓基质细胞可分化为中胚层组织细胞，实验予进一步探讨体外分离培养的骨髓基质细胞向神经干细胞分化的可能性，以及是否能继续定向分化为神经细胞及神经胶质细胞，为神经系统疾病细胞移植治疗提供种子细胞。
方法：实验于2007-02/09在泸州医学院神经生物学研究室进行。①动物：选择5只普通级SD大鼠，由泸州医学院实验动物中心提供，实验过程中对动物的处置符合动物伦理学标准。②实验方法：大鼠戊巴比妥钠腹腔麻醉，取双侧胫骨和股骨，磷酸盐缓冲液冲洗骨髓腔，采用密度梯度离心法从大鼠骨髓中分离培养骨髓基质细胞，胰蛋白酶与EDTA联合消化，传至第4代，用含20 μg/L碱性成纤维细胞生长因子、20 μg/L表皮生长因子、N2辅助因子的DMEM/F12培养液向神经干细胞诱导分化。③实验评估：观察原代、传代培养及诱导分化后的骨髓基质细胞生长情况和形态变化，采用SABC法进行免疫细胞化学检测神经细胞特异性标志的表达。
结果：①骨髓基质细胞形态观察：原代细胞接种1 d后开始贴壁增殖，3 d后多数贴壁，贴壁细胞呈梭形或扁平形；10 d后90%细胞融合，以长梭形为主，突起粗大，形成网状、片状；传代后细胞贴壁速度加快，增殖能力更强，7 d左右达到融合。②诱导分化后细胞生长情况和形态变化：第4代骨髓基质细胞向神经干细胞诱导分化7 d，成球的细胞脱离瓶底，悬浮在细胞液中。将细胞离心弃上清，换成血清分化液后，细胞球逐渐贴壁，球周围很快发出突起，分化为星形胶质细胞样细胞、神经元样细胞及少突胶质细胞样细胞。③神经细胞特异性标志的表达：骨髓源性细胞球表达巢蛋白，呈棕黄色，为神经干细胞；从细胞球分化的细胞抗胶质纤维酸性蛋白、微管相关蛋白2及半乳糖脑苷脂均呈阳性。
结论：骨髓基质细胞能在体外诱导分化出神经干细胞，且骨髓源性神经干细胞可进一步定向分化为神经细胞及神经胶质细胞。
关键词：骨髓基质细胞；神经干细胞；分化

高小青，杜杰，杨朝鲜，吴岩，邓莉，袁琼兰.大鼠骨髓基质细胞体外培养向神经干细胞的诱导分化[J].中国组织工程研究与临床康复，2008，12(12):2305-2308 [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2305(ps).pdf]

Differentiation of rat bone marrow stromal cells into neural stem cells in vitro

Abstract

AIM：It has been confirmed that bone marrow stromal cells (BMSCs) can differentiate into mesoblastemic tissue cells. This experiment further investigated the possibility of differentiation of BMSCs in rats into neural stem cells in vitro, and into neural cells and neuroepithelial cells, so as to provide seed cells for cell transplantation of nervous system disease.

METHODS: Experiments were performed at the Department of Neurobiology, Luzhou Medical College from February to September 2007.①Five common SD rats were selected from the Experimental Animal Center of Luzhou Medical College. To handle animals in experiment was accorded with ethical standards. ②The rats were anesthetized with napental. Bone marrow was obtained from the femoral and tibial bones by washing with phosphate buffered solution. BMSCs from rats were isolated by combining density gradient centrifugation, digested with trypsin and EDTA. The fourth passage of BMSCs were induced by DMEM/F12 medium containing 20 μg/L epidermal growth factor (EGF), 20 μg/L basic fibroblast growth factor (bFGF) and N2 factor. ③The growth and the morphological changes of primary, passaged cells and induced BMSCs were observed. Specific markers of neural cells were identified by SABC immunocytochemisty staining.

RESULTS: ①Morphological observation of BMSCs: Firstly inoculated cells began to adhere and
proliferate in 1 day. Three days later, most of them attached to the bottom and showed fusiform or flat shape. 90 % cells were confluence on the 10th day. Most cells showed like fusiform with thick and long apophyses that connected each other into network. Generated BMSCs rapidly adhered to wall, and proliferated speed of subculture cells was faster than that of primary culture cells. On the 7th day, subculture cells confluence. ②The growth and the morphological changes of induced BMSCs: The 4th passage BMSCs were induced to neural stem cells. About 7 days, cell clusters attached previously broke away the bottom, floated and became dissociative spheres. Cells were centrifugalized and supernatant was removed. These spheres gradually attached to the wall and became differentiated when they were placed in serum medium, radially protruding processes from the spheres, then they were differentiated into astrocyte-like, neuron-like oligodendrocyte-like cells. ③Expression of specific markers of neural cells: Bone marrow-derived spheres expressed nestin, and differentiated cells from spheres expressed glial fibrillary acidic protein (GFAP), microtubule-associated protein（MAP2）and galactocerebroside (Galc).

CONCLUSION: BMSCs can be induced into neural stem cells and further neural stem cells derived from bone marrow can differentiate into neural cells and neuroglial cells.

Gao XQ, Du J, Yang CX, Wu Y, Deng L, Yuan QL.Differentiation of rat bone marrow stromal cells into neural stem cells in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2305-2308(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2305(ps).pdf]