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目的建立人退变髓核组织细胞体外培养体系,探索最佳培养条件,为髓核退变机制研究和组织工程研究筛选种子细胞。方法采用酶消化法分离人退变髓核组织细胞,进行单层培养,通过CellCountingKit-8评估细胞活性,采用免疫细胞化学染色等方法比较不同代次细胞形态结构及软骨样表型和增殖活性。结果成功进行细胞单层培养和鉴定,P1～P3代细胞活性和Ⅱ型胶原的表达有明显降低(P<0.05)。结论 P1、P2和P3代细胞表型保持符合软骨样细胞特征,能够为髓核退变体外研究和组织工程研究提供种子细胞。
Objective To establish an in vitro culture system of degenerated nucleus pulposus cells and to explore the best culture conditions for screening degeneration mechanism of nucleus pulposus and tissue engineering screening of seed cells. Methods Human degenerated nucleus pulposus cells were isolated by enzymatic digestion and cultured in monolayer. CellCountingKit-8 was used to assess cell viability. Immunocytochemical staining was used to compare the morphology, cartilage-like phenotype and proliferative activity of different generations of cells. Results The cell monolayer culture and identification were successfully performed. The cell viability and the expression of type Ⅱ collagen in P1 ~ P3 were significantly decreased (P <0.05). Conclusion The cell phenotypes of P1, P2 and P3 are in line with the characteristics of chondrocytes and can provide seed cells for in vitro research and tissue engineering of degenerative nucleus pulposus.