Studies on the mechanism of endochondral ossification using a clonal chondrocyte like cell line newly established from a human chondrosarcoma - approach using the methods of cellular and molecular biology

1. HCS-2/8细胞同时产生IGF-I和IGF-II，并表达这些生长因子的mRNA。这些细胞有I型和II型IGF受体。IGF-I和igf - II都刺激了蛋白多糖的合成。这些发现表明，这两种IGFs在维持HCS-2/8细胞中高水平的蛋白多糖合成中起自分泌分化因子的作用。与之前使用其他细胞系的研究结果相反，我们发现HCS-2/8细胞上的igf -II型受体在igf -II刺激蛋白多糖合成的信号转导中发挥重要作用。碱性FGF仅在稀疏培养下刺激HCS-2/8细胞增殖，且细胞产生大量bFGF，提示bFGF过表达参与了细胞系的永久生长。tgf -刺激了细胞内DNA和蛋白多糖的合成。该因子支持HCS-2/8细胞在无血清培养基中的增殖和分化。通过northern blotting和RT-PCR，我们发现HCS-2/8细胞表达了许多与软骨细胞分化相关的基因，如聚集蛋白核心蛋白、II型胶原、X和XI型胶原、IGF-I和IGF-II以及这些受体和维生素D_3受体。抗坏血酸诱导碱性磷酸酶活性及其mRNA表达。它还能诱导细胞肥大。没有检测到以下原癌基因的转录本：c-sis， c-met, c-src, c-lyn, c-fgr, c-ros， c-pim和Blym。然而，12个其他原癌基因（int-2、erbB、c-abl、c-raf-1、c-fyn、K-ras、H-ras、c-mos、c-myc、c-myb、c-fos和c-jun）的转录本很容易被Northern分析检测到。两种原癌基因c-fos和c-raf-1在过融合阶段的转录本水平分别高于培养早期阶段的10倍和3倍，这表明与肥大有关。

1. HCS-2/8 cells produced both IGF-I and IGF-II and expressed mRNA of these growth factors. The cells had types I and II receptors for IGF. Both IGF-I and II stimulated proteoglycan synthesis. These findings suggest that both IGFs act as autocrine differentiation factors in maintaining a high level of proteoglycan synthesis in HCS-2/8 cells. In contrast to previous findings using other cell lines, we found that IGF-type II receptor on HCS-2/8 cells play an important role in signal transduction by IGF-II-stimulated proteoglycan synthesis.2. Basic FGF stimulated the proliferation of HCS-2/8 cells only in sparse culture and the cells produced large amount of bFGF, suggesting that overexpression of bFGF is involved in permanent growth of the cell line. TGF-beta stimulated DNA and proteoglycan syntheses in the cells. The factor supports the proliferation and differentiation of HCS-2/8 cells in serum-free medium.3. Using northern blotting and RT-PCR, we found that HCS-2/8 cells express many genes related to chondrocyte differentiation such as aggrecan core protein, collagen types II, X and XI, IGF-I and IGF-II and these receptors and vitamin D_3 receptor. Ascorbic acid induced alkaline phosphatase activity and its mRNA expression. It also induced hypertrophy of the cells.4. There were no detectable transcripts for the following proto-oncogenes: c-sis, c-met, c-src, c-lyn, c-fgr, c-ros, c-pim, and Blym. However, transcripts of 12 other proto-oncogenes (int-2, erbB, c-abl, c-raf-1, c-fyn, K-ras, H-ras, c-mos, c-myc, c-myb, c-fos, and c-jun) are readily detectable by Northern analysis. Two proto-oncogenes, c-fos and c-raf-1 had an elevated levels of transcripts as high as 10 and 3 folds, respectively, at the overconfluent phase in comparison with the earlier phases of culture, suggesting relation to hypertrophy.