成人骨髓间质干细胞诱导分化为神经元样细胞及其与细胞分裂的关系

Relationship between mitosis and differentiation from human mesenchymal stem cell into neuron-like cell

目的探讨成人骨髓间质干细胞(hMSC)向神经元样细胞分化过程中分化与细胞分裂的关系。方法从成人骨髓中分离骨髓间质干细胞(MSC),培养扩增。用参芪液诱导MSC分化为神经元样细胞,经免疫组化鉴定其神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。诱导后进行神经元样细胞计数,掺入Brdu测定,计算标记系数。采用双标记间接免疫荧光法检测神经元样细胞特异性表面标记(NSE、NF)与细胞分裂的标记Brdu的关系。抑制DNA合成,计算Brdu、NSE和NF阳性细胞数。结果MSC经参芪液诱导后,可见神经元样细胞。免疫组化显示诱导出的神经元样细胞NSE、NF阳性,GFAP阴性。MSC诱导分化后的细胞总数增加,诱导分化后12h内细胞增殖较快。30%以上分化的神经元样细胞在表达NSE和NF的同时可见Brdu标记,而另一部分分化的神经元则无Brdu标记。抑制DNA合成,并不会阻止神经元的分化,仍有70%的分化神经元表达神经元特异性标记蛋白NSE。结论MSC诱导分化的部分神经元样细胞可进行细胞分裂,但抑制细胞分裂不影响神经元分化。

Objeetive To study the relationship between mitosis and differentiation in the process of induction from human mesenchymal stem cells （hMSC） into neuron-like cells. Methods Mesenchymal stem cells （MSC） were separated from human marrow, cultured, and expanded in culture medium. After MSC was induced to differentiate into neuron-like cells with Shenqi Liquid, expression of neuron-specific enolase （NSE）, neurofilament （NF）, and glial fibrillary acidic protein （GFAP） were detected by immunocytochemistry, respectively. Total cell count before and after differentiation were calculated, the Brdu labeling index （LI. the ratio of Brdu-labeled to total cells） was measured. Using double label indirect immunofluorescence, the relationship between neuron-like cells and mitosis was illuminated. Results MSC exhibited neuronal phenotype after treated with inducers, Shenqi Liquid. The expressions of NSE and NF in the neuron-like cells were positive, but GFAP was not found. The number of total cells was increased, especially during the first 12 h of induction. Over 30% neuron expressed NSE and NF incorporated Brdu, while another part of neuron not incorporated Brdu. Inhibiting the DNA synthesis, hMSC still differentiated into neuron-like cells and over 70% expressed neuron specific gene products such as NSE and NF. Conelusion Some neuron-like cells after induction would undergo mitosis, but inhibiting the DNA synthesis does not influence the neurogenesis.