Making a DNA Chip for the Study of Cartilage, and a New Challenge for the Repair and Regeneration of Cartilage Tissue.

本研究采用基因捕获法和cDNA差减法对软骨特异表达基因进行了全面的鉴定。此外，商业上可获得的cDNA芯片也用于分析。虽然我们最初为该项目开发的组织特异性基因捕获系统尚未完成，但系统构建的副产品信号序列特异性基因捕获载体已经很好地鉴定了软骨细胞产生的细胞表面和细胞外基质分子。类似地，我们发现cDNA消减分析通过适当地选择组织或细胞用于基因表达的比较而工作良好。例如，我们通过抑制性消减杂交比较了关节软骨和生长板软骨的基因表达，并且可以鉴定出在关节软骨中特异性表达而在生长板软骨中不表达的几个基因。一种这样的分子是润滑素，其特异性地表达在关节软骨的表层上，并且对于关节的润滑具有重要功能。对于其他分子，我们可以通过北方印迹分析证实它们在每个软骨中的特异性表达。然而，使用市售的cDNA微阵列分析并没有给我们带来我们预期的丰硕成果。这是由于商业上可获得的cDNA芯片对于分析软骨中表达的基因仍然是不完善的。因此，全面分析软骨组织中的基因表达仍然是非常重要的，而软骨特异性基因表达谱芯片的制备是我们面临的最紧迫的挑战。

In this research project, gene trap and cDNA subtraction methods were used to identify comprehensively the genes specifically expressed in cartilage. Furthermore, commercially available cDNA chip was also used for the analysis. Although tissue specific gene trap system that we have been originally developing for this project has not been completed yet, signal sequence specific gene trap vector, a byproduct of the system construction, has been working well to identify cell surface and extracellular matrix molecules produced by chondrocytes. Similarly, we found that cDNA subtraction analysis works well by selecting tissues or cells properly for a comparison of gene expression. For example, we compared the gene expression of articular cartilage and growth plate cartilage by suppression subtractive hybridization, and could identify several genes specifically expressed in articular cartilage but not in growth plate cartilage. One such molecule is lubricin that is specifically expressed on the superficial layer of articular cartilage, and has an important function for the lubrication of joint. Of the other molecules, we could confirm their specific expression in each cartilage by Northern blot analysis. However, cDNA microarray analysis using commercially available one did not give us fruitful results as we expected. This is due to the fact that commercially available cDNA chips are still imperfect for analyzing genes expressed in cartilage. Therefore, comprehensive analysis of genes expressed in cartilage is still very important, and the production of cartilage specific cDNA microarray is most urgent challenge for us.