人颈椎间盘髓核细胞的体外培养和鉴定

Cultivation and identification of human cervical nucleus pulposus cells

目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。

Objective：To establish the system for culturing the human cervical nucleus pulposus cells and to identify the phenotype.Methods The human cervical nucleus pulposus cells were isolated by collagenase digestion method and cultured in monolayer by culture solutions of DF12＋20%FBS.Morphologic changes and growth of the cells were detected by microscope.The cell cycle and apoptosis rate were detected by Flow cytometric.The morphological structure and the phenotype were identified with the toluidine blue staining and immunocytochemistry means.Result The apoptosis rate of primary cells was 6.1±1.4%.The Cells at S phase were 7.3± 0.5%.The cells were polygonal or short wedged morphology.The second passage cells grew more fast than that of the primary cells.The cells displayed intense toluidine blue metachromasia.The cells expressed collagen type Ⅱ,but only a few elliptic gaint cells expressed CK8.Conclusion The human cervical nucleus pulposus cells were isolated and cultured in monolayer successfully.A few cells of the nucleus pulposus still maintain notochord cells phenotype in adult specimens.