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

本课题采用基因诱捕法和cDNA减法对软骨中特异性表达的基因进行综合鉴定。此外，还使用市售cDNA芯片进行分析。虽然我们最初为本项目开发的组织特异性基因陷阱系统尚未完成，但作为系统构建的副产品，信号序列特异性基因陷阱载体在识别软骨细胞产生的细胞表面和细胞外基质分子方面已经取得了很好的效果。同样，我们发现cDNA减法分析通过选择合适的组织或细胞来比较基因表达效果很好。例如，我们通过抑制减法杂交比较了关节软骨和生长板软骨的基因表达，发现了几个在关节软骨中特异性表达而在生长板软骨中不特异性表达的基因。其中一种分子是润滑素，它在关节软骨的浅层上特异性表达，对关节的润滑有重要作用。其他分子，我们可以通过Northern blot分析确认它们在每个软骨中的特异性表达。然而，使用市售的cDNA微阵列分析并没有像我们预期的那样给我们带来丰硕的结果。这是由于商业上可用的cDNA芯片在分析软骨中表达的基因方面仍然不完善。因此，全面分析软骨中表达的基因仍然非常重要，而制作软骨特异性cDNA微阵列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.