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

我用胰岛素培养atdc5细胞，并使其分化为软骨细胞。达到融合后，这些细胞进一步生长，形成阿利新蓝染色的软骨样结节。结节于融合后2周检出，逐渐增多至融合后4周。然而，即使在融合后4周，非软骨组织仍存在于结节间区。在达到融合2周后，我将培养基换成含有维生素C的α - mem，继续培养2周，然后诱导钙化组织。加入β -甘油磷酸酯（2-5mM）可稳定诱导钙化。接下来，我在达到合流后的第0w、1w、2w、3w分别用^<35>S04的^ 3h -氨基葡萄糖进行代谢标记，并用凝胶过滤和离子交换色谱分析合成的蛋白聚糖结构。结果表明，该细胞可合成大的软骨型蛋白聚糖（aggrecan型）和小的非软骨型蛋白聚糖（Decorin、Biglycan型）。特别是，即使在达到融合3周后，培养基中也存在相当数量的非软骨型蛋白多糖，而在细胞层中，软骨型蛋白多糖只存在。这些结果提示软骨结节的内层形成软骨组织，而软骨结节的表层仍存在非软骨组织，这一概念与本研究的组织学观察相一致。综上所述，ATDC5细胞作为软骨形成的模型是合适的，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.