TGF-β3,HA,PTHrP对人脐带间充质干细胞成软骨分化的影响

Effects of transforming growth factor β3, hyaluronic acid, and parathyroid hormone-related protein on chondrogenic differentiation of human umbilical cord mesenchymal stem cells

目的探讨TGF-β3、透明质酸(hyaluronic acid,HA)、甲状旁腺激素相关蛋白(parathyroid hormonerelated protein,PTHrP)对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUC-MSCs)成软骨分化的影响,优化培养条件的组合方式。方法体外分离培养人脐带间充质干细胞,形态学观察,流式检测表面抗体及成脂、成骨分化鉴定。取P3代细胞,加入不同组合的细胞因子进行成软骨诱导培养,分为1~21 d TGF-β3(G1)、1~28 d TGF-β3(G2)、1~21 d TGF-β3/1~21 d HA(G3)、1~28 d TGF-β3/1~28 d HA(G4)、1~21 d TGF-β3/1~21 d HA、14~21 d PTHrP(G5)、1~28 d TGF-β3、1~28 d HA、14~28 d PTHrP(G6)、1~28 d TGF-β3、1~28 d HA、21~28 d PTHrP(G7) 7组。结果诱导培养28 d和21 d相比较,TGF-β3组和TGFβ3/HA组COL2α1、COL10α1、SOX9、ACAN的表达量上升;TGFβ3/HA/PTHrP组COL2α1、SOX9、ACAN的表达量上升,COL10α1下降。培养相同天数时,TGFβ3/HA组较TGF-β3组COL2α1、SOX9、ACAN的表达量上升;TGFβ3/HA/PTHrP组同TGF-β3/HA组比较,COL2α1、ACAN、SOX9的表达量增加,COL10α1表达量下降,且PTHrP加入2周较1周效果更明显。差异均有统计学意义。HE和阿利新蓝染色结果与实时荧光定量PCR的结果一致。结论 TGF-β3、HA、PTHrP可以不同程度的促软骨分化,其中联合应用TGF-β3,HA 28 d,在第14天时加入PTHrP一起培养,这种组合方式促进HUC-MSCs成软骨分化效果最好,且能抑制细胞肥大。

Objective To investigate the effects of transforming growth factor (TGF)-β3, hyaluronic acid (HA), and parathyroid hormone-related protein (PTHrP) on chondrogenic differentiation of human umbilical cord mesenchymal stem cells and to optimize the combination of culture conditions. Methods Human umbilical cord mesenchymal stem cells were isolated and cultured in vitro for morphological examination, surface antibody detection, and adipogenic and chondrogenic differentiation. Different combinations of cytokines were added to P3 generation cells for chondrogenic differentiation as follows: TGF-β3 on days 1-21 (G1), TGF-β3 on days 1-28 (G2), TGF-β3 on days 1-21 + HA on days 1-21 (G3), TGF-β3 on days 1-28 + HA on days 1-28 (G4), TGF-β3 on days 1-21 + HA on days 1-21 + PTHrP on days 14-21 (G5), TGF-β3 on days 1-28 + HA on days 1-28 + PTHrP (G6) on days 14-28, and TGF-β3 on days 1-28 + HA on days 1-28 + PTHrP on days 21-28 (G7). Results The expression levels of COL2α1, COL10α1, SOX9, and ACAN were higher in the TGF-β3 and TGF-β3/HA groups at 28 than 21 days of culture.In addition, the expression levels of COL2α1, SOX9, and ACAN were higher and that of COL10α1 was lower in the TGF-β3/HA/PTHrP group. When cultured at the same times, the expression levels of COL2α1, SOX9, and ACAN were higher in the TGF-β3/HA group than TGF-β3 group, and the expression levels of COL2α1, ACAN, and SOX9 were higher and that of COL10α1 was lower in the TGF-β3/HA/PTHrP group than TGF-β3/HA group. The effect of PTHrP addition for 2 weeks was significantly more obvious than that for 1 week ( P <0-05). The results of hematoxylin-eosin and alcian blue staining were consistent with the results of real-time quantitative polymerase chain reaction. Conclusions TGF-β3, HA, and PTHrP can promote chondrogenic differentiation to different extents. When TGF-β3 and HA were combined for 28 days and PTHrP was added on the 14th day (G6), this combination promotes best the chondrogenic differentiation of human umbilical cord mesenchymal stem cells and inhibits the cell hypertrophy.