不同来源椎间盘髓核间质干细胞特性及其多向分化能力研究

Multiple differentiation potential of mesenchymal stem cells in nucleus pulposus of different sources： a comparative study

目的比较来源于先天性脊柱侧凸与腰椎间盘退变髓核组织中髓核间质干细胞增殖特性及其多向分化能力差别。方法收集2例先天性脊柱侧凸与2例腰椎间盘突出患者的椎间盘组织，采用酶消化法分离细胞，在含体积分数为10％胎牛血清的低糖DMEM完全培养液中培养髓核间质干细胞，用MTT法及台盼蓝染色检测脊柱侧凸与椎间盘退变来源的两组细胞的代谢活力与增殖能力。流式细胞仪鉴定两组髓核间质干细胞CD44、CD105、CD29、CD24表达差异，观察两组细胞成骨、成脂、成软骨分化能力。结果侧凸组髓核间质干细胞以梭形为主，退变组髓核间质干细胞以多边形及三角形为主，传代后两组细胞大多以纺锤梭形为主。MTT及台盼蓝染色法提示侧凸组代谢活力及增殖能力强于退变组。侧凸组和退变组髓核间质干细胞CD44、CD105、CD29表达比例分别为97％～100％和88．7％～97％，CD24表达均为阴性，两者均不表达造血细胞标志物CD45、CD34、CD14、HLA—DR。侧凸组与退变组髓核间质干细胞均可向成骨、成软骨分化，但均未向成脂分化。结论先天性脊柱侧凸与椎间盘退变髓核组织中均存在髓核间质干细胞，前者的髓核间质干细胞具有较强的细胞代谢活性及增殖能力，两者成骨、成软骨分化能力无差别，但均无成脂能力。

Objective To differential the biologic characteristics and multiple differentiation potential of mesenchymal stem cells in nucleus pulposus in （NP-MSC） scoliosis patient and patient with degenerative intervertbral disc. Methods The human nucleus pulposus-mesenchymal stem cells were isolated and cultured with enzyme digestion from 2 patients of scoliosis and 2 patient with degenerative intervertbral disc separately. Cellular proliferation was detected with MTT assay and trypan blue. The immunophenotype expression of NP-MSC was detected by flow cytometry in scoliosis and degenerative group. The multiple differentiation ability of cells was assessed respectively using alizarin red dye, Oil red O dye and immunohistochemical staining. Results The primary cell morphology of scoliosis NP-MSC was the shape of spindle, while the degenerative NP-MSC was inhomogeneous. However, both of them became spindle shape after passages. The scoliosis NP-MSC was stronger than degenerative one in metabolic activity and proliferation. The percentage of positive antigen expression of CD44, CD105 and CD29 was 97%-100% in scoliosis NP-MSC group, but 88.7%- 97% in the degenerative group. The expression of mature cell marker CD24 was negative in both groups. Furthermore, the MSC isolated from both groups differentiated along the osteogenic, chondrogenic but not adipogenic lineages. Conclusion The scoliosis and degenerative NP contains mesenchymal stem cells. Moreover the scoliosis NP-MSC had stronger ability of cell metabolic activity and proliferation. These cells have multiple differentiation potential, with the exception of their adipogenic differentiation ability.