人骨髓间充质干细胞与髓核细胞共培养后的类髓核分化效应及其对髓核细胞表型的调节作用

Co-culture induces human bone marrow-derived mesenchymal stem cell differentiation and modulation of the nucleus pulposus cell phenotype

目的探讨人骨髓间充质干细胞分别与正常及退变髓核细胞共培养后的类髓核分化效应及其对髓核细胞表型的调节作用并进行比较。方法取来源于腰椎退变患者以及脊柱侧弯患者的间盘组织,通过改良Pfirrmann法对其退变程度进行分级,进行髓核细胞(NPCs)的分离培养及鉴定,通过术中椎弓根钉道取血,进行骨髓间充质干细胞(BMSCs)的分离培养及鉴定。取传至第3代细胞进行共培养。建立5个培养组:BMSCs单独培养组,退变NPCs单独培养组,正常NPCs单独培养组,BMSCs与退变NPCs共培养组,BMSCs与正常NPCs共培养组。共培养7 d后,RT-PCR法检测各组细胞SOX-9、聚集蛋白聚糖Aggrecan与Ⅱ型胶原蛋白的mRNA表达。结果相较于BMSCs单独培养组,共培养组BMSCs的SOX-9、Aggrecan与Ⅱ型胶原蛋白的mRNA表达均有明显增加(P<0.05)。同时与BMSCs与正常NPCs共培养组相比,BMSCs与退变NPCs共培养组BMSCs的SOX-9、Aggrecan与Ⅱ型胶原蛋白的mRNA表达均有明显增加(P<0.05)。与退变NPCs单独培养组相比,共培养组退变NPCs的SOX-9、Aggrecan与Ⅱ型胶原蛋白的mRNA表达均明显增加(P<0.01)。与正常NPCs单独培养组相比,共培养组正常NPCs的SOX-9、Aggrecan与Ⅱ型胶原蛋白的mRNA表达均明显增加(P<0.01)。结论 BMSCs与NPCs共培养时的相互作用能促使BMSCs向类髓核表型分化,较之正常NPCs,退变NPCs与BMSCs共培养时能更明显地促进BMSCs向类髓核表型分化。同时BMSCs对NPCs的调节作用能促进其特征性相关基因表达,与退变NPCs共培养时,这一效应依然明显。

Objective To investigate the interaction between human bone marrow-derived mesenchymal stem cells （BMSCs） and nucleus pulposus cells （NPCs） from both nondegenerate and degenerate discs during in vitro co-culture, and make a comparison. Methods The isolation and identification of BMSCs and NPCs were performed on bone marrow blood and nucleus pulposus from either disk degeneration patients or scoliosis patients. Five groups were designed as follows： A group （BMSCs group）, B group （degenerated NPCs group）, C group （nondegenerated NPCs group）, D group （BMSCs with degenerated NPCs co-cultured group） and E group （BMSCs with nondegenerated NPCs co-cultured group）. The mRNA expressions of collagen Ⅱ, SOX-9 and aggrecan of these two types of cells were assessed using quantitative real-time PCR after 7 days. Results Compared with A group, the mRNA expressions of collagen Ⅱ, SOX-9 and aggrecan significantly upregulated in D and E groups. Compared with E group, the mRNA expression was upregulated in D group （P 〈 0.05）. Compared with B group, the mRNA expression of D group increased （P 〈 0.01）. Compared with C group, the mRNA expression of E group also increased （P 〈 0.01）. Conclusion During co-culture process, the interaction between BMSCs and NPCs can stimulate BMSCs and differentiate to a NP-like phenotype; Compared with nondegenerate NPCs, co-culture with degenerate NPCs can more significantly stimulate BMSCs differentiating to a NP-like phenotype. At the same time, BMSCs can regulate both nondegenerate and degenerate NPCs.