Freyria A M, Ronzière M C, Roche S, Rousseau C F, Herbage D
Institut de Biologie et Chimie des Protéines, CNRS-UPR, 69367 Lyon Cedex 07, France.
J Cell Biochem. 1999 Nov;76(1):84-98. doi: 10.1002/(sici)1097-4644(20000101)76:1<84::aid-jcb9>3.0.co;2-z.
Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.
软骨细胞的表型表达可通过改变培养技术以及诸如维生素和生长因子等试剂在体外进行调节。我们研究了抗坏血酸、视黄酸（0.5和10微摩尔）以及二氢细胞松弛素B（3、10、20微摩尔二氢细胞松弛素B）单独或联合使用（抗坏血酸+视黄酸或抗坏血酸+二氢细胞松弛素B），对在含有10%胎牛血清和各种试剂的培养基中高密度培养长达4周的胎牛骨骺软骨细胞成熟诱导的影响。在没有任何试剂的情况下或单独使用视黄酸或二氢细胞松弛素B时，细胞的代谢活性在第6天后仍然非常低，没有诱导I型或X型胶原合成，碱性磷酸酶活性也没有增加。用新鲜抗坏血酸处理的软骨细胞分别在6天和13天后显示出与I型和X型表达相关的活跃蛋白质合成。这种成熟并未伴随着细胞明显肥大或高碱性磷酸酶活性。在抗坏血酸处理的培养物中添加视黄酸会降低II型胶原合成水平，并延迟I型和X型胶原的诱导，这两种胶原仅在30天后才出现。在抗坏血酸和最高剂量视黄酸（10微摩尔）同时存在的情况下，观察到碱性磷酸酶活性显著增加（15至20倍）。二氢细胞松弛素B也是抗坏血酸处理诱导成熟的有效抑制剂，因为软骨细胞保持其圆形形状并合成II型胶原，而没有诱导I型或X型胶原。通过二维凝胶电泳在所有培养条件下比较了蛋白质分泌模式。二维凝胶电泳在整个研究过程中观察到的分泌蛋白质模式的重要定性和定量变化证实了抗坏血酸、视黄酸和二氢细胞松弛素B对软骨细胞分化的不同调节作用。
