Cloning and functional analysis of novel genes related to chondrocyte differentiation using the human chondrocytic cell lines

为了分离出在软骨内成骨过程中发挥重要作用的新的功能分子和调控分子，我们首先对新建立的人软骨肉瘤细胞系（HCS）进行了表征，并建立了兔生长软骨细胞增殖分化的模型培养体系。然后，我们使用差异显示pcr分析了HCS细胞系、正常兔软骨细胞和其他类型细胞中mrna的表达。因此，我们获得了30种hcs特异性软骨细胞的DNA片段。测定了30种HCS细胞中17种的核苷酸序列。碱基序列比对发现了7个新的序列标记和少数与已知DNA序列同源的序列标记。其中一个序列标签（标签号）。24)与结缔组织生长因子（CTGF）核苷酸序列具有高度的结构同源性，相应基因（hcs24）在培养的HCS细胞和兔生长软骨细胞中选择性表达，而在培养的骨肉瘤细胞的成骨细胞中不表达。tgf - β或BMP-2可上调HCS细胞中hcs24的表达。在兔生长软骨细胞体外培养过程中，其表达量在体内早期增生软骨细胞相应阶段达到最大值。原位杂交结果显示，hcs24仅在胚胎小鼠肋软骨和脊柱增生性软骨细胞中表达。反义寡核苷酸强烈抑制HCS细胞的增殖，增加其蛋白多糖的合成。反义寡聚物还能提高培养兔生长软骨细胞碱性磷酸酶活性。这些结果表明Hcs24蛋白是由肥大软骨细胞产生的，它促进生长软骨细胞的增殖并抑制其向软骨内成骨的分化。

To isolate new functional and regulatory molecules, which play an important role in the process of endochondral ossification, we first characterized the newly established human chondrosarcoma cell lines (HCS) and established a model culture system on the proliferation and differentiation of rabbit growth cartilage cells. We then analyzed mRNAs expressed in HCS cell lines, normal rabbit chondrocytes and other types of cells using differential display-PCR.Consequently, we obtained 30 species of chondrocyte- of HCS-specific DNA fragments. Nucleotide sequences of 17 of 30 species derived from HCS cells were determined. Comparison of the base sequences revealed seven novel sequence tags and a few sequence tags showing homology with known DNA sequences. One of the sequence tags (tag no.24) showed high structural homology with the nucleotide sequence of connective tissue growth factor (CTGF) and the corresponding gene (hcs24) was selectively expressed in HCS cells and rabbit growth cartilage cells in culture but was not expressed in osteoblastic cells of osteosarcoma cells in culture. The expression of hcs24 in HCS cells was up-regulated by the addition of TGF-beta or BMP-2. During in vitro culture of rabbit growth cartilag cells, its expression reached a maximum at the stage corresponding to early hypertrophic chondrocytes in vivo. In situ hybridization revealed that hcs24 was expressed only in the hypertrophic chondrocytes of costal cartilage and the vertebral column in embryonic mice. Anti-sense oligonucleotides strongly inhibited the proliferation of HCS cells and increased their proteoglycan synthesis. The anti-sense oligomer also increased alkaline phosphatase activity in rabbit growth cartilage cells in culture. These results suggest that Hcs24 protein is produced by hypertrophic chondrocytes and that it promotes the proliferation of growth cartilage cells and suppresses their differentiation toward endochondral ossification.