三磷酸腺苷对骨髓间充质干细胞向软骨细胞分化的影响及机制研究

Effect and Mechanism of Adenosine Triphosphate on Differentiation of Bone Marrow Mesenchymal Stem Cells into Chondrocytes

目的探讨三磷酸腺苷(Adenosine triphosphate,ATP)对骨髓间充质干细胞(BMSCs)向软骨细胞分化的影响及其作用机制。方法取4~5周龄SD大鼠的BMSCs,体外培养至第3代。以PNPP法检测不同浓度ATP(150μmol/L、300μmol/L、600μmol/L)及BBG(P2X7受体抑制剂)对BMSCs碱性磷酸酶表达的影响。采用RT-PCR和real time PCR法检测ATP及BBG对BMSCs多个成软骨诱导指标mRNA表达的影响。结果加入ATP后,BMSCs碱性磷酸酶表达明显增加(P<0.05),其中ATP浓度为150μmol/L和300μmol/L时碱性磷酸酶表达量最高(P<0.05);多个成软骨指标,如SOX6、SOX9及COL2A1等的表达水平提高(P<0.05);加入P2X7受体抑制剂后,ATP对碱性磷酸酶的促进作用降低(P<0.05),成软骨指标SOX6、SOX9及COL2A1等的表达亦明显减少(P<0.05)。结论一定浓度的ATP对BMSCs向软骨细胞分化具有促进作用,可能是通过介导P2X7受体而发挥作用。

Objective To investigate the effect of adenosine triphosphate on the differentiation of BMSCs into chondrocytes and its mechanism.Methods The primary cells of BMSCs from 4-5 weeks old SD rats were cultured to the third generation in vitro.PNPP method was used to detect the effects of ATP with different concentrations(150,300 and 600μmol/L)and BBG(receptor inhibitor of P2X7)of on alkaline phosphatase expression in BMSCs.The mRNA expression of multiple cartilage markers was detected by semi-quantitative reverse transcription polymerase chain reaction and real time PCR.Results After ATP was added,the expression of alkaline phosphatase in BMSCs was significantly increased(P<0.05).The highest expression of alkaline phosphatase was observed when ATP concentration was 150 and 300μmol/L(P<0.05).The expression levels of indicators such as SOX6,SOX9 and COL2A1 were increased(P<0.05).After the addition of P2X7 receptor inhibitor,the promotion effect of ATP on alkaline phosphatase decreased(P<0.05),and the expressions of chondrogenic indicators such as SOX6,SOX9 and COL2A1 were also significantly decreased(P<0.05).Conclusion A certain concentration of ATP can promote the differentiation of bone marrow mesenchymal cells into chondrocytes,which may play a role through the mediation of P2X7 receptor.