The Role of proteoglycans in chondrocyte differentiation of mesenchymal stem cells

1.评价超声对骨髓间充质干细胞向软骨细胞分化的影响。培养于微球中的人MSCs经转化生长因子-β(TGF-β)处理后，经阿新蓝染色和琼脂糖凝胶免疫染色鉴定为软骨细胞，但未处理的细胞微球和单独BMP-6处理的细胞微球不能分化为软骨细胞。此外，每天对经转化生长因子-β处理的细胞颗粒施加20min的低强度超声，可促进软骨细胞的分化。这些结果提示，转化生长因子-β对颗粒间充质干细胞向软骨细胞的分化是必需的，超声促进了其分化。使用软骨细胞N1511细胞，我们评价了Verscan/PG-M在软骨细胞分化中的作用。N1511细胞早期表现为一过性高表达，晚期表现为聚集素的高表达，反映了其在软骨细胞分化过程中的体内表达模式。当使用反义技术抑制verscan的表达时，根据实时荧光RT-PCR的评估，随后的aggrecan的表达被减少和延迟。这些结果表明，在软骨细胞分化的早期阶段，瞬时的verscan表达对软骨细胞的分化具有正向调节作用。除了高瞬时表达外，Verscan还持续表达，特别是在关节软骨中。我们发现Verscan与透明质酸和链接蛋白在那里是共定位的。生化分析进一步证实了凡西嘉聚集体的存在。这些结果表明存在两个蛋白多糖聚合体，它们可能起着不同的作用。

1. We evaluated the effects of ultrasound (US) on differentiation of mesenchymal stem cells (MSCs) toward chondrocyte snarl cartilage matrix formation. When human MSCs cultured in the pellets were treated with transforming growth factor-beta (TGF-b), they differentiated into chondrocytes as assessed by alcian blue staining and immunostaining for aggrecan, but non-treated cell pellets and cell pellets treated with BMP-6 alone did not. Furthermore, when low-intensity US was applied for 20 min everyday to the TGF-beta-treated cell pellets, chondrocyte differentiation was enhanced. These results suggest that TGF-b is essential for chondrocyte differentiation of MSCs in pellets and US enhances the differentiation.2. Using chondrogenic/chondrocytic N1511 cells, we evaluated the role of versican/PG-M in chondrocyet differentiation. N1511 cells exhibited transient high expression of versican at an early stage and that of aggrecan at a late stage, which reflects their in vivo expression patterns during chondrocyte differentiation. When versican expression was inhibited using an antisense technology, the following aggrecan expression was decreased and delayed, as assessed by realtime RT-PCR. These results suggest that transient versican expression at the early stage positively regulate chondrocyte differentiaion.3. In addition to high transient expression, versican is continuously expressed, especially in the articular cartilage. We found that versican is colocalized with both hyaluronan and link protein there. Biochemical analysis further confirmed the presence of versican aggregate. These results suggest the presence of tow proteoglycan aggregates, which may play distinct roles.