Investigation of the mechanism of proliferation and differentiation of cartilage tissue using chondrocytes derived from p53-/-mouse.

1)免疫组化和RT-PCR分析表明，EP 2是关节软骨细胞中PGE 2的主要受体。EP 2特异性激动剂诱导MMA 2中cAMP的剂量依赖性增加，MMA 2是来源于p53-/-小鼠关节软骨的软骨细胞系。比较EP 2激动剂处理前后MMA 2基因表达谱的差异，共鉴定出22个EP 2激动剂上调表达的基因，其中包括多个促生长和促炎基因。在治疗与EP 2激动剂，人关节软骨细胞表现出增加BrdU的掺入，和大鼠股骨器官培养显示关节软骨细胞中的PCNA染色增加。这些结果表明，PGE 2信号通过EP 2促进关节软骨细胞的生长，EP 2激动剂有望成为治疗软骨退行性疾病的新药物。2）生长板钙化相关因子的分离。MMR 14和MMR 17的基因表达谱，其中前者在单层培养中产生钙化结节，而后者不产生钙化结节。比较，以分离生长板钙化过程中涉及的因素。几个细胞粘附分子，细胞外基质，信号分子和转录因子基因被分离，作为差异表达，基因，包括OB-钙粘蛋白。OB-钙粘蛋白在mRNA和蛋白水平的表达在MMR 14中随着时间的推移而增加，但在MMR 17中未观察到表达。在肋骨生长板中，OB-cadherin在钙化软骨中表达，提示其参与了生长板软骨细胞的最终分化步骤。

1) Investigation of PGE2 signalImmunohistochemical and RT-PCR analysis showed that the EP2 is the major receptor for PGE2 in articular chondrocytes. EP2 specific agonist induced a dose-dependent increase of cAMP in MMA2, which is a chondrocyte cell line derived from articular cartilage of p53-/-mice. Gene-expression profile of MMA2 before and after the treatment with EP2 agonist was compared, and 22 genes up-regulated genes by EP2 agonist were identified including several growth-promoting and apoptotis-inhibiting genes. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of BrdU, and the organ culture of rat femur showed an increase in PCNA staining in articular chondrocytes. These results suggested that PGE2 signal through EP2 enhances the growth of articular chondrocytes, and the EP2 agonist is a candidate for a new therapeutic compound for the treatment of cartilage degenerative disease.2) Isolation of factors involved in the calcification in growth plate.Gene expression profiles of MMR14 and MMR17, of which the former but not the later produced the calcified nodules in monolayer culture, was compared to isolate the factors involved in the process of calcification in the growth plate. Several cell-adhesion molecule, extracellular matrix, signal molecule and transcriptional factor genes were isolated, as differentially expressed, genes including the OB-cadherin. Expression of OB-cadherin both at mRNA and protein levels increased with times in MMR14 in the monolayer culture, but no expression was observed in MMR17. In the growth plate of rib, OB-cadherin was detected in calcifying cartilages, suggesting its involvement of the step of final differentiation of growth plate chondrocytes.