Signal transmission of TGF-beta superfamily in chondrocyte

从关节重建的临床方面，我们研究了如何修复关节面。利用培养的软骨细胞进行组织工程修复关节是关节修复的方法之一，目前已取得显着进展，利用培养的软骨细胞修复关节，重要的问题是软骨细胞的特性，尤其是其作为关节软骨细胞的表型。换句话说，希望所使用的软骨细胞永不骨化。鉴于软骨细胞的这些特性，我们研究了TGF-β超家族在软骨细胞中的信号传递。在本研究中，我们研究了佛波醇，12-肉豆蔻酸酯，13-乙酸酯（PMA），一种有效的和特异性的蛋白激酶C（PKC）激活剂和Staurosporine，一种有效的非选择性的PKC抑制剂，从13日龄鸡胚胸骨软骨细胞的分化的影响，以及PKC是否作为其作用的介导剂。硫酸化蛋白多糖（PG）和I型和II型胶原蛋白被用作分化的标志物。免疫印迹法检测PKC表达。PMA呈剂量依赖性（1-50 ng/ml）抑制软骨形成，其作用可能是通过PKC从胞内转位到细胞膜而介导的。星形孢菌素增强软骨形成（1- 80 nM），并以剂量依赖性方式引起总PKC耗竭。已经观察到，响应于星形孢菌素（4和80 nM），N-单甲基，L-精氨酸（NMA）（一氧化氮合酶（NOS）的特异性抑制剂）对PG合成的协同刺激，以及对NO合成的相应抑制。PMA对PG合成的影响不受NMA的调节，提示NO可能不参与PMA抑制鸡胸骨软骨细胞分化的作用，但星形孢菌素可能至少部分通过NO依赖的机制促进软骨细胞的分化

From a clinical aspect of joint reconstruction, we have investigated how to repair the joint surface. One of the methods to repair the joint is tissue engineering using cultured chondrocytes which has developed markedly.To repair the joint using cultured chondrocytes, important problem is character of chondrocyte, especially their phenotype as articuler chondrocytes. In other words, it is desirable that the used chondrocytes never ossifies. Considering these character of chondrocytes, we investigated signal transmission of TGF-beta superfamily in chondrocytes. In the present study we examined the effect of phorbol, 12-myristate, 13-acetate (PMA), a potent and specific protein kinase C(PKC) activator and Staurosporine, a potent nonselective PKC inhibitor, on the differentiation of sternal chondrocytes from 13-day-old chick embryo, and whether PKC functions as a mediator of their effects. Sulfated proteoglycan (PG) and type I and type II collagen were used as markers of differentiation. PKC expression was assayd by immunoblotting. PMA inhibited chondrogenesis dose-dependently (1-50 ng/ml), and its effects were possibly mediated by the translocation of PKC from the cytozol to the membrane. Staurosporine enhanced chondrogenesis (1-80nM) and caused the depletion of total PKC in a dose-dependent fashion. A synergistic stimulation of PG synthesis by N-monomethyl, L-arginine (NMA), a specific inhibitor of nitric oxide synthase (NOS), in response to Staurosporine (4 and 80 nM), with a corresponding suppression of NO synthesis, has been observed. The effect PMA on PG synthesis was not modulated by NMA.We suggest that NO may no be involved in the inhibition of chick sternal chondrocyte differentiation by PMA,but that Staurosporine may promote chondrogenic differentiation at least in part through a NO-dependent mechanism