培养软骨细胞再形成生长板样软骨组织的研究

REORGANIZATION OF GROWTH-PLATE-LIKE TISSUE BY ISOLATED CHONDROCYTES IN CULTURE

分离的家兔肋软骨的生长板软骨细胞在含10%的胎牛血清的IMDM培养液中进行高密度培养。培养的软骨细胞经过增殖、分化重新构建形成了生长板样软骨组织;在组织学上,细胞的排列呈现明显的方向性,肥大细胞位于上侧、增殖细胞位于下面从而形成明显的细胞分化区带,即增殖、成熟区和肥大区,并且细胞纵向排列成柱状;在生化代谢方面,软骨细胞DNA,蛋白多糖和碱性磷酸酶的合成具有动态的严格的时间规律性,与在体生长板软骨细胞分化的进程相一致,完整地模拟了在体软骨细胞分化及其代谢的全部过程。

Growth-plate cartilage is organized into four cellular zones containing resting, proliferating, maturing, and hypertrophic cells. Rabbit chondrocytes were isolated from growth-plate costal cartilage of 4-week-old New Zealand rabbits, the cells (15×104) were suspended in 1 ml of Iscove's modified Dulbecco's medium (IMDM) with 10% fetal bovine serum, 50 μg ascorbic acid, and 60 μg kanamycin (medium A), then transferred to a 15ml of plastic centrifuge tube, and centrifuged at 1500 rpm for 5 min. The cell pellet was incubated at 37℃ under 5% CO2 in air. The cultures reorganized into growth plate-like tissue which could be seen 7-14 days after cell seeding. This growth-plate, histologically, was organized longitudinally into cellular columns and horizontally into four cellular zones containing resting, proliferating, maturing and hypertrophic cells. The hypertrophic cells in the upper were large in size and round or oval in shape, the proliferating and the mature chondrocytes in the lower were small in size and spherical or elongated in shape. These chondrocytes were surrounded by an extensive matrix. Biochemically, DNA content of cultures began to rise on the 2nd day after cell seeding and reached a plateau after 10 days later. The uronic acid content increased from day 4 and reached the maximum on day 15.In contrast in the early culture, alkaline phosphatase activity was extremely low, it began to rise on day 9 and was the highest on day 20.The sequential increase of DNA, uronic acid and alkaline phosphatase contents was analogous to the in vivo changes of growth-plate chondrocytes.