WNT激活剂促进人骨髓基质干细胞向软骨细胞分化的研究

The effects of WNT activator on chondrocytogenesis of human bone marrow stromal cells

目的 观察WNT激活剂SB-216763是否具有促进人骨髓基质干细胞（hMSCs）向软骨细胞分化的功能.方法 6例hMSCs,将成软骨细胞诱导分化的hMSCs与SB-216763共同培养,光镜下计数Alcian blue染色阳性的软骨岛的数目、以Alcian blue染色法定量检测由hMSCs分化而来的软骨细胞分泌硫酸化糖胺聚糖的能力,以逆转录-聚合酶链反应（RT-PCR）检测软骨细胞标志基因SOX9、Collagen Ⅱ、Aggrecan的表达变化.结果 在hMSCs成软骨诱导分化过程中,SB-216763可以显著增加软骨岛的数目（133.17±16.58比9.67±1.87,P〈0.01）,显著增加hMSCs分化的软骨细胞分泌硫酸化糖胺聚糖的量（1.6～2.6倍,P〈0.01）,并促进SOX9、Collagen Ⅱ和Aggrecan基因的表达.结论 SB-216763可以促进体外hMSCs向软骨细胞的分化和成熟.

Objective To investigate the effects of a WNT signaling pathway activator, SB-216763,on chondrocytogenesis of human bone marrow stromal cells （hMSCs） in vitro. Methods Fresh bone marrow samples were obtained with institutional review board （IRB） approval as discarded femoral tissue. Low-density mononuclear cells were isolated by density centrifugatian on Ficoll/Histopaque 1077. Adherent hMSCs were expanded 2 passages. Upon near-confluence of hMSCs ,medium was changed to α-MEM with chondrogenic supplements （1% ITS^＋1, 10^-8 mol/L dexamethasone,50 mg/L ascorbate-2-phosphate, 40 mg/L proline,100 mg/L pyruvate） with or without 5 μmoL/L SB-216763. After 10 days,the cultures were stained with 0.1% alcian blue in 3% acetic acid （pH=1.0） for sulfated glycosaminoglycans and the number of alcian-blue-positive nodules was counted with 4×magnification, hMSCs from five women and one man, aged 57-77 years,wero used to measure content of sulfated glycesaminoglycans by absorbance （A）. After 14 days, the effects of SB-216763 on the expression of chondrocyte signature genes were quantified with reverse transeription polymerase chain reaction （RT-PCR）. Results During the process of induction differentiation of hMSCs into chondrocytes ,SB-216763 could produce more cartilage nodules （133.17±16.58） than the control group （9.67±1.87 ） （ P〈0.01）. SB-216763 stimulated the production of sulfated glycosaminogly-cans by 1.6 to 2.6-fold as indicated by alcian blue staining （P〈0.01）. Consistent with the matrix data, analysis of chondrocyte signature genes showed that the expression of SOX9, collagen Ⅱ, and aggrecan was enhanced by 5 μmol/L SB-216763. Conclusion The WNT signaling pathway activator,SB-216763 stimu-lates in vitro chondrocytogenesis and cartilage matrix production by hMSCs.