课题背景：2006年山东省自然科学基金资助项目。课题取得了下列研究成果：①通过全骨髓贴壁法获得较纯化的骨髓间充质干细胞，并成功诱导分化为神经干细胞。②实验证实BrdU（5-溴脱氧尿嘧啶）可作为神经干细胞体内示踪的理想标记物。

应用要点：①通过全骨髓贴壁法分离纯化骨髓间充质干细胞，获取了较量多数量的细胞，并经过表皮生长因子和碱性成纤维细胞生长因子诱导后，细胞Nestin、NSE、GFAP表达阳性，表明在适当的条件下骨髓间充质干细胞可以诱导分化出神经干细胞。②为了解决神经干细胞在实验研究中的示踪问题，采用BrdU对神经干细胞进行体外和体内标记，1周后其标记率高达85%以上，说明BrdU标记和检测的准确性高、标记率高，是跟踪监测神经干细胞移植的理想标记物。

相关链接：对神经干细胞的研究近年来许多实验均致力于神经干细胞来源的问题，以期获得一个可靠的获取大量纯化神经干细胞的方法，其中最受关注的是骨髓间充质干细胞，骨髓间充质干细胞可以来源于自体,并且获取简单，从而克服了许多异体神经干细胞移植的弊端，为神经干细胞治疗带来新的希望。但是要将骨髓间充质干细胞真正应用于临床,仍然存在一些问题：①需要进一步检测从骨髓间充质干细胞分化而来的神经干细胞是否具有神经干细胞的一系列功能。②需要深入了解调控骨髓间充质干细胞向神经干细胞转化的分子机制等。

摘要
目的：来源于骨髓间充质干细胞的神经干细胞是最近研究的一个热点。实验拟进一步验证通过全骨髓贴壁法分离纯化骨髓间充质干细胞并诱导分化为神经干细胞的效率，并对其的体内外标记进行了观察。
方法：实验于2006-11/2007-05在青岛大学医学院附属医院脑血管病研究所完成。①实验材料：选取出生4周左右Wistar大鼠，雌雄不拘，SPF级，体质量200 g左右，由青岛市实验动物和动物实验中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：选用出生4周左右Wistar大鼠，由其股骨和胫骨分离纯化骨髓间充质干细胞，加碱性成纤维细胞生长因子和表皮生长因子诱导其分化为神经干细胞，并进行免疫组织化学鉴定。以5-溴脱氧尿嘧啶核苷（BrdU）标记神经干细胞，在24孔板中进行细胞免疫组织化学和免疫荧光鉴定。将标记的神经干细胞定位注入大鼠基底节区, 1周后取脑作石蜡切片, 进行免疫组织化学和免疫荧光染色。
结果：获得了较纯化的骨髓间充质干细胞，经诱导72 h后，部分细胞增殖分裂成小圆形,呈团簇状半悬浮状态。继续培养 72 h可见细胞呈现典型神经元样改变，细胞伸出两个或多个突起, 胞体呈多角形或不规则形, 折光性较强, 细胞突起之间相互连接成网状,可见细胞核及核仁，经诱导后有巢蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白阳性细胞表达。采用BrdU掺入DNA合成期的神经干细胞，1周后体内外标记阳性率> 85%。
结论：采用全骨髓贴壁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神经干细胞。BrdU可作为神经干细胞体内示踪的理想标记物。
关键词：骨髓间充质干细胞；神经干细胞；5-溴脱氧尿嘧啶核苷；诱导分化

张建富 ，孟庆海，金澎，刘广义.全骨髓贴壁法获纯化骨髓间充质干细胞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):2240-2244
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2240(ps).pdf]

Differentiation of bone marrow mesenchymal stem cells into neural stem cells by whole bone marrow adherence method

Abstract

AIM：The loss of neurons is the severest in the basal forebrain of senile dementia patients. This study was aimed to verify the effects of neural stem cells (NSC) transplantation on the parvalbumin (PV)-positive neurons in the basal forebrain and the abilities of leaning and memory in the rat model of senile dementia.

METHODS: This study was done in the Department of Anatomy, Guangzhou Medical College from December 2005 to July 2006.①Animals: SPF class adult male SD rats (n=28) were randomly divided into three groups: normal control group (n =8), model control group (n =8), and NSC group (n =12). Furthermore, new-born SD rats (n =10) were used to isolate and culture NSC. All the manipulations were in accordance with the animal ethnic standard.②Methods: The rats of model group and NSC group were made by left fimbria-fornix transaction. NSCs were obtained from the basal forebrain by using the serum-free culturing. And 4 μL NSC suspension (2.5×1010 L-1) was stereotaxically implanted into the injured lesion (coordinates: anterior-posterior, +0.6 mm; lateral, +0.6 mm; dorsal-ventral, -5.5 mm from bregma) instantly after fimbria-fornix transaction.③Evaluation: Four weeks postoperation, the learning and memory abilities were measured by Y-maze test. Then, the brains were taken out after anesthesia and cut on a microtome. The number of PV-positive neurons in the basal forebrain was analyzed by using ABC immunohistochemical staining method and image analysis.

RESULTS: ①Effects on learning and memory abilities: The abilities in NSC group were significantly higher than that in model control group (P < 0.05 or P < 0.01), and almost reached the normal level (P > 0.05).②Effects on PV-positive neurons: Compared with normal control group, PV-positive neurons in the medial septum (MS) and the vertical limb of the diagonal band of Broca (VDB) in model control group decreased by 62.5% and 30.4%, respectively. The decrease extent in NSC group was distinctly smaller than that in model control group (P < 0.01). The area, circumference, and gray value of PV-positive neurons in MS and VDB of model control group were significantly reduced compared with normal control group (P < 0.05 or P < 0.01). However, all the characteristic parameters in NSC group were significantly increased than those in model control group (P < 0.05 or P < 0.01).③Correlative analysis: The number of PV-positive neurons in MS and VDB was negatively correlated with the times of learning of rats (r=-0.76 to -0.79, P < 0.01) and positively correlated with the ability of memory (r=0.78 to 0.84, P < 0.01), which were measured by Y-maze test.

CONCLUSION: NSC transplantation can supply and protect PV-positive degenerative neurons in the basal forebrain and is effective on improving the abilities of learning and memory in the rat model of senile dementia. The relation of them is positive correlative.

Gu HG, Long DH, Li XB, Zhang GP, Su T, Li JM, Leng SL. Effect of neural stem cells transplantation on parvalbumin-positive neurons of the basal forebrain and abilities of learning and memory in a rat model of senile dementia.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2235-2239(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2235(ps).pdf]