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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。
BACKGROUND: The potential of bone marrow mesenchymal stem cells and hematopoietic stem cells to differentiate into neurons has been agreed. In addition, other researchers think that in addition to bone marrow mesenchymal stem cells and hematopoietic stem cells, they also host CXCR4 positive tissue-oriented stem cells, including skeletal muscle, heart, liver and nerve tissue-oriented stem cells. OBJECTIVE: The experiment intends to further confirm that the nerve tissue-oriented stem cells reside in the bone marrow and seek to establish a new method for the isolation and culture of the nerve tissue-oriented stem cells. Design: Open experiment. Unit: Department of Anatomy, Second Military Medical University. Materials: The adult rat SD rats were provided by Animal Center of PLA Second Military Medical University. DMEM / F12, B27, N2 were purchased from Invitrogen; bFGF from CytoLab Ltd; EGF from Invitrogen; Nestin, CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein and other primary antibody rabbit Santa Cruz anti- (Clone11-5B), CNPase (Clone AP20) were NeoMarkers mouse anti-rat monoclonal antibody; Fluorescence (FITC, Cy3) labeling kit was purchased from Wuhan Boster Biological Engineering Co., Ltd .; immunohistochemistry Kits were purchased from Vector Laboratories. NycoPrepTM Separation Fluid (1.077A) was purchased from Axis-Shield Company. METHODS: The experiment was performed at the Institute of Tissue Engineering, Second Military Medical University of Chinese PLA from January 2004 to December 2006. The bilateral femur and tibia of SD rats were taken and the bone marrow cavity was washed. The mononuclear cell layer was separated by NycoPrepTM separation liquid. DMEM / F12 (1: 1) serum-free neural stem cell culture medium containing 2% B27, g / L basic fibroblast growth factor, 20 μg / L epidermal growth factor, 1 × 105 U / L penicillin, 100 mg / L streptomycin) were isolated from the bone marrow by first adherent and post- Cultivate monoclonal growth of nerve tissue-oriented stem cells. MAIN OUTCOME MEASURES: CXCR4 and nestin proteins were detected by immunocytochemistry in NSCs and neurons in glial cells and markers of glial cells. RESULTS: ①Nervous tissue - derived stem cell spheres expressed NSC marker nestin and tissue - oriented stem cell marker protein CXCR4. ②Nerve tissue-oriented stem cell spheres were naturally differentiated in DMEM medium containing 15% fetal bovine serum. The differentiated cells were spindled or polygonal with 2-5 cell protrusions. Immunofluorescence showed that the neuronal marker protein β- tublinIII and MAP2ab, as well as the glial marker CNPase and glial fibrillary acidic protein positive. Conclusion: Nerve tissue-oriented stem cells exist in the bone marrow and can naturally differentiate into neuron-like cells and glial-like cells.