Biochemical and histological analysis of proteoglycans systhsized by cartilaginous cell line, ATDC5.

用胰岛素诱导ATDC 5细胞分化为软骨细胞。细胞融合后进一步生长，形成软骨样结节，阿辛蓝染色。在融合后2周检测到结节，并逐渐增加，直到融合后4周。融合后4周，非软骨组织仍残留于结节间区。而在达到融合后2周，我将培养基更换为含有维生素C的α-MEM并继续培养2周，然后诱导钙化组织。然后在<35>细胞融合后的0、1、2、3周分别用^3 H-Glucosamine中的^SO4进行代谢标记，用凝胶过滤和离子交换层析分析合成的蛋白多糖的结构。结果表明，该细胞合成大的软骨型蛋白聚糖（聚集蛋白聚糖型）和小的非软骨型蛋白聚糖（Decorin，双糖蛋白聚糖型）。特别是在细胞融合后3周，培养液中仍有大量的非软骨型蛋白多糖存在，而细胞层中仅存在软骨型蛋白多糖。结果表明，软骨组织形成于软骨结节的内层，而非软骨组织仍存在于软骨结节的表层，这一观点与本研究的组织学观察相一致，ATDC 5细胞是一种合适的软骨形成模型，但在分析培养基成分时需要注意。

I cultured ATDC 5 cells with insulin, and made these cells differentiate into chondrocytes. After reaching confluency, these cells grew further and made cartilage-like nodules stained with alcian blue. The nodules were detected on 2 weeks after reaching confluency and gradually increased until 4 weeks after confluent. However, even on 4 weeks after confluent, non-cartilage tissue still remained internodullar area. While, on 2 weeks after reaching confluency, I changed the medium into alpha-MEM with Vitamine C and further cultured for 2 weeks, then calcified tissue was induced. By adding beta-glycerophosphate (2-5mM), the calcification was stably induced.Next, I tried matabolic labeling with ^<35>S04 of ^3H-Glucosamine on 0w, 1w, 2w, 3w after reaching confluency, and analyzed the synthesized proteoglycan structure with gel filtration and ion exchange chromatography. The results indicate that large cartilage type proteoglycan (aggrecan type) and small non-cartilage type proteoglycan (Decorin, Biglycan type) were synthesized by this cell. Especially, even on 3 weeks after reaching confluency, considerable amount of non-cartilage type proteoglycan existed in culture medium, while in cell layr, cartilage type proteoglycan exclusively existed. These results suggested that cartilage tissue is formed inner layr of cartilage nodules and non-cartilage tissue still remains on the surface layr of cartilage nodules, and this concept was conssisted with histological observations in this study.As a conclude, ATDC5 cell is appropriate as a model of chondrogenesis, but some attentions are needed when analyzing medium component.