【摘要】 目的:目前多采用胎牛血清培养骨髓间充质干细胞,实验应用自体血清培养骨髓间充质干细胞并诱导其向神细胞分化,拟验证其可行性及并分析其可能的机制。方法:实验于2005-10/2006-08在同济医学院附属同济医院神经外科实验室完成。培养骨髓间充质干细胞所用骨髓来源为本院需行骨髓穿刺的健康成人(共3例,年龄26～44岁),本人及家属知情同意。梯度离心法分离成人骨髓间充质干细胞,并利用自体血清进行体外培养扩增。主要观察指标:采用相差显微镜观察细胞形态变化;透射电镜观察骨髓间充质干细胞的超微结构;流式细胞术鉴定细胞表面标记物;3～5代细胞利用生长因子和N2添加剂进行诱导分化,采用RT-PCR检测Netin和神经元特异性烯醇化酶mRNA在诱导前后的表达。结果:①细胞形态变化:利用自体血清培养的骨髓间充质干细胞主要呈长或宽扁的梭形,在体外传代扩增速度较快。②超微结构:电镜下细胞表面可见细胞核较大,不规则,核质比较小,胞质中有丰富的细胞器,可见缝隙连接。③细胞表面标记物鉴定:多数细胞为CD44、CD105阳性,CD34、CD45阴性。④Netin和神经元特异性烯醇化酶mRNA的表达:诱导后部分细胞呈神经元样改变,Nestin mRNA表达水平于3d达高峰,随后减少,神经元特异性烯醇化酶mRNA于5～7d达高峰,并可持续数日。结论:应用自体血清可成功培养骨髓间充质干细胞,并在体外条件下定向诱导分化为神经元样细胞。更多还原

【Abstract】 AIM: Mesenchymal stem cells (MSCs) are cultured with fetal calf serum mostly at present. Autologous serum is utilized to culture MSCs and induce MSCs into neural cells. This article is aimed to investigate the probability and mechanisms of using autologous serum. METHODS: The experiment was performed at the Laboratory of Department of Neurosurgery,Tongji Hospital,Tongji Medical College from October 2005 to August 2006. Bone marrow was collected from three healthy adults aged 26-44 years who would receive bone marrow aspiration at this hospital. All patients and their family members singed the informed consent. MSCs were separated from human bone marrow by gradient centrifugation and expanded with autologous serum in vitro. The morphology of the MSCs was studied with the phase-contrast microscope and the transmission electron microscope. Surface markers of cultured cells were evaluated by flow cytometry. 3-5 passage cells were induced by growth factor and N2. The changes of Nestin and neuronspecific enolase (NSE) mRNA expressions were detected by RT-PCR before and after induction. RESULTS: ①Most of the human MSCs were long or flat fusiform shape and expanded rapidly in vitro. ②The ultrastructural features of the MSCs were larger abnormal shaping nucleus,decreased nuclear-cytoplasmic ratio,gap junctions and affluent organelles. ③CD44 and CD105 were positive in most cells while CD34 and CD45 were negative. ④After induced neuron-like cells were observed. Nestin mRNA expressed mostly after 3 days and decreased gradually while NSE mRNA mostly expressed after 5-7 days and lasted for several days. CONCLUSION: Autologous serum cultured MSCs can be induced into neuron-like cells in vitro.更多还原