Abstract



BACKGROUND: It has been widely accepted that both bone marrow-derived mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have the capacity to differentiate into neural lineages. Some scholars believe that in addition to HSCs and MSCs, bone marrow (BM) also harbors a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue.
OBJECTIVE: To make sure that neural tissue-committed stem cells (NTCSCs) reside in the bone marrow, and to establish a purification and culture method for bone marrow-derived NTCSCs.
DESIGN: Opening animal study.
SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA.
MATERIALS: Adult Sprague-Dawley (SD) rats (pathogen-free) were provided by the Animal Center of the Second Military Medical University of Chinese PLA. Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor (EGF, Invitrogen Company), basic fibroblast growth factor (bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company), mouse anti-rat microtubule associated protein 2ab (MAP2ab) (Clone11-5B), cyclic nucleotide 3'phosphohydrolase （CNPase, Clone AP20, NeoMarkers Company）, fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co., Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole(DAPI)(Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.
METHODS: This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to December 2006. Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27,1% N2, 20 μg/L bFGF, 20μg/L EGF, 1×105 U/L penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium.
MAIN OUTCOME MEASURES: NTCSCs were identified by immunocytochemistry for CXCR4, a marker of TCSCs and nestin, a marker of neural stem cells, and neural lineages marker protein after differentiation of cellular spheres.
RESULTS: The NTCSCs spheres expressed nestin, a neural stem cell marker as well as CXCR4, a marker of TCSCs. The NTCSCs' spheres were naturally differentiated in DMEM medium with 15% fetal bovine serum. The differentiated cells expressed β-Tublin Ⅲ, MAP2ab, CNPase and GFAP, markers of neural lineages.
CONCLUSION: NTCSCs reside in bone marrow and naturally differentiate into neural lineages in vitro.

Department of Anatomy, Second Military Medical University of Chinese PLA, Shanghai 200433, China

Zhang Zhi-ying☆, Doctor, Associate professor, Department of Anatomy, Second Military Medical University of Chinese PLA, Shanghai 200433, China
zhiying1963@163.
com

Correspondence to: Zhang Chuan-sen, Doctor, Department of Anatomy, Second Military Medical University of Chinese PLA, Shanghai 200433, China Chuansen@yahoo.
com.cn

Received:2007-11-17
Accepted:2007-12-04
(07-50-10-6366/H)

Zhang ZY, Ren CL, Zhang CS, Li L, Dang RS, Kong ZD.Identification of neural tissue-committed stem cells, a new cellular group, in bone marrow.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):
1577-1580(China)

[www.zglckf.com/
zglckf/ejournal/
upfiles/08-8/
8k-1577(ps).pdf]

骨髓中一类新细胞群——神经组织定向
干细胞的确立☆

摘要
背景：骨髓间充质干细胞和造血干细胞具有分化为神经类细胞的潜能已得到共识。另有研究者认为骨髓中除骨髓间充质干细胞和造血干细胞以外，还寄居着CXCR4阳性的组织定向干细胞，包括骨骼肌、心、肝及神经组织定向干细胞。
目的：实验拟进一步证实神经组织定向干细胞寄居于骨髓，并寻求确立神经组织定向干细胞分离、培养的新方法。
设计：开放性实验。
单位：解放军第二军医大学解剖学教研室。
材料：所用成年清洁级SD大鼠由解放军第二军医大学动物中心提供。DMEM/F12，B27，N2均购自Invitrogen公司；bFGF源于CytoLab Ltd；EGF源于Invitrogen公司；Nestin、CXCR4，β-Tublin Ⅲ，神经胶质纤维酸性蛋白等一抗为Santa Cruz公司兔抗鼠多克隆抗体；MAP2ab（Clone11-5B），CNPase（Clone AP20）为NeoMarkers公司小鼠抗大鼠单克隆抗体；荧光（FITC，Cy3）标记试剂盒购自武汉博士德生物工程有限公司；免疫组化试剂盒购自Vector Laboratories公司。NycoPrepTM分离液（1.077A）购自Axis-Shield 公司。
方法：实验于2004-01/2006-12在解放军第二军医大学组织工程研究所完成。取SD大鼠双侧股骨及胫骨，冲洗骨髓腔，经NycoPrepTM分离液分离单核细胞层，DMEM/F12（1∶1） 无血清神经干细胞培养液（内含2%B27，1%N2，20 μg/L碱性成纤维细胞生长因子， 20 μg/L 表皮生长因子,1×105 U/L青霉素, 100 mg/L链霉素），通过先贴壁、后悬浮的方法从骨髓中分离、培养单克隆生长的神经组织定向干细胞。
主要观察指标：通过免疫细胞化学法检测神经组织定向干细胞细胞球CXCR4和nestin蛋白，以及细胞球自然分化后神经元以及神经胶质细胞的标志蛋白。
结果：①神经组织定向干细胞细胞球表达神经干细胞标志蛋白nestin和组织定向干细胞标志蛋白 CXCR4。②神经组织定向干细胞细胞球在含15%胎牛血清的DMEM培养液中可自然分化，分化细胞呈梭形或多角形，有2~5个细胞突起，免疫荧光检测显示神经元标志蛋白β-tublinIII及MAP2ab，以及神经胶质标志蛋白CNPase和神经胶质纤维酸性蛋白阳性。
结论：骨髓中存在神经组织定向干细胞，可自然分化为神经元样细胞及神经胶质样细胞。
关键词：骨髓；神经组织定向干细胞；细胞培养；鉴定；细胞移植

张志英，任丛莉，张传森，李 亮，党瑞山，孔征东
解放军第二军医大学解剖教研室，上海市 200433
张志英☆，女，1963年生，山西省平遥县人，汉族，2002年上海中医药大学毕业，博士，副教授，主要从事神经再生与修复的研究。
通讯作者：张传森, 博士，解放军第二军医大学解剖教研室，上海市 200433

中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01577-04
张志英，任丛莉，张传森，李亮，党瑞山，孔征东.骨髓中一类新细胞群——神经组织定向干细胞的确立[J].中国组织工程研究与临床康复，2008，12(8):1577-1580
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1577(ps).pdf]
（Edited by Martin F/Song LP/Wang L）