大鼠BMSCs条件培养基对NSCs形态、生长及分化的影响

The Effect of Bone Mesenchymal Stem Cells Culture Medium on the Morphology, Growth and Differentiation of Neural Stem Cells

通过对SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)及新生鼠神经干细胞(neural stem cells,NSCs)进行体外分离、培养及鉴定后,观察BMSCs条件培养基对NSCs向神经细胞分化的影响。采用全贴壁培养法培养BMSCs,并采用流式细胞术鉴定其特异性表面抗原标记。无血清技术培养NSCs,采用免疫荧光技术鉴定其特异性抗原标记。BMSCs条件培养基(含10%胎牛血清的DMEM培养基)对NSCs诱导7 d后,镜下观察细胞形态和生长,并采用免疫荧光技术检测NSCs分化。BMSCs高表达CD90、CD29(>90%),而CD45呈阴性表达。免疫荧光染色显示,NSCs标记蛋白Nestin、SOX2为阳性。NSCs经BMSCs培养液诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为73.80%和50.47%;NSCs经胎牛血清(fetal bovine serum,FBS)诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为42.14%和31.90%。BMSCs条件培养基可诱导NSCs分化为神经元及星形胶质细胞,其中BMSCs分泌的细胞因子在诱导分化中可能发挥重要作用。

This study was designed to observe the effect of BMSCs（bone mesenchymal stem cells） conditioned medium on the differentiation of NSCs（neural stem cells） after the isolation, culture and identification of SD rat BMSCs and NSCs. BMSCs were cultured using method of all-adherent culture and identified with specific antigen marker by flow cytometry. NSCs were cultured using serum-free method and identified with specific antigen marker by immunofluorescence. NSCs were induced with BMSCs conditioned medium（DMEM medium containing 10% fetal bovine serum） for 7 d, and then we could observe morphology of NSCs and detect NSCs differentiation using immunofluorescence technique. We found that BMSCs highly expressed CD90, CD29（〉90%）, whereas CD45 expression was rarely; and immunofluorescence showed that specific marker of NSCs such as Nestin, SOX2 was positive reaction. The positive rate of expression MAP-2 and GFAP were 73.80% and 50.47% respectively after NSCs were induced with BMSCs conditioned medium for 7 d. Meanwhile, after NSCs had been induced and differentiated by fetal bovine serum（FBS） for 7 d, immunofluorescence identification indicated that both MAP-2 and GFAP were positive as well, with the rates 42.14% and 31.90%, respectively. BMSCs conditioned medium can induce NSCs to differentiate into neurons and astrocytes, in which cytokines secreted by BMSCs maybe play the vital role.