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

1)PGE_2信号的研究免疫组织化学和RT-PCR分析表明，EP2是关节软骨细胞PGE_2的主要受体。EP2特异性激动剂诱导P53-/-小鼠关节软骨细胞系MMA2中cAMP含量呈剂量依赖性增加。比较EP2激动剂作用前后MMA2细胞的基因表达谱，共鉴定出22个受EP2激动剂上调的基因，其中包括几个促进生长和抑制细胞凋亡的基因。在EP2激动剂作用下，人关节软骨细胞BrdU掺入增加，大鼠股骨器官培养显示关节软骨细胞增殖细胞核抗原染色增加。这些结果表明，PGE2信号通过EP2促进关节软骨细胞的生长，EP2激动剂是治疗软骨退行性疾病的新的候选药物。2)生长板钙化相关因子的分离。比较MMR14和MMR17在单层培养中产生钙化结节的基因表达谱，以分离生长板中参与钙化过程的因子。分离到了一些细胞黏附分子、细胞外基质、信号分子和转录因子等差异表达基因，其中包括OB-钙粘蛋白。在单层培养的MMR14中，OB-cadherin在mRNA和蛋白水平的表达均随培养时间的延长而增加，而在MMR17中未见表达。在肋骨生长板中，钙化软骨中检测到OB-钙粘蛋白，提示其参与了生长板软骨细胞的终末分化过程。

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.