Xu C, Oyajobi B O, Frazer A, Kozaci L D, Russell R G, Hollander A P
Department of Human Metabolism, Sheffield University Medical School, United Kingdom.
Endocrinology. 1996 Aug;137(8):3557-65. doi: 10.1210/endo.137.8.8754787.
The aim of this study was to investigate the effects of insulin-like growth factor-I, transforming growth factor-beta (TGF-beta), and interluekin-1 alpha (IL-1 alpha) on the deposition and degradation of a cartilage-like matrix in high-density pellet cultures of adult bovine chondrocytes. Proteoglycan was determined by toluidine blue staining and colorimetric assay. Type II collagen was determined by immunohistochemical staining and its unwinding in situ by a recently developed immunoassay. Bovine nasal chondrocytes cultured as pellets deposited a well-organized extracellular matrix of proteoglycan and type II collagen. Insulin-like growth factor-I (2-10 ng/ml) increased the synthesis and incorporation into the matrix of both these proteins. TGF-beta (2-10 ng/ml) also increased proteoglycan synthesis. However it inhibited proteoglycan deposition, presumably through increased degradation of the molecule, as shown by increased release of aggrecan fragments into the tissue culture medium. TGF-beta had no effect on type II collagen deposition. In pellet cultures of bovine nasal or articular chondrocytes, 20 ng/ml IL-1 alpha induced a significant degradation of both proteoglycan and type II collagen. The effect on collagen clearly involved proteolytic cleavage of its triple helix because there was an increase in the proportion of unwound type II collagen in the matrix, as well as a loss of total type II collagen. In explant cultures of intact bovine articular cartilage, incubation with 50 ng/ml IL-1 alpha stimulated significant degradation of the proteoglycan but no degradation of the type II collagen. These results demonstrate that although the articular chondrocytes are capable of degrading type II collagen when isolated, they do not do so in situ, presumably because of some inherent property of the mature extracellular matrix. This study demonstrates the utility of pellet cultures when investigating chondrocyte-mediated turnover of cartilage matrix and its modulation by cytokines and growth factors.
本研究旨在探讨胰岛素样生长因子-I、转化生长因子-β（TGF-β）和白细胞介素-1α（IL-1α）对成年牛软骨细胞高密度微团培养中软骨样基质沉积和降解的影响。通过甲苯胺蓝染色和比色测定法测定蛋白聚糖。通过免疫组织化学染色及其在原位通过最近开发的免疫测定法解旋来测定II型胶原蛋白。作为微团培养的牛鼻软骨细胞沉积了组织良好的蛋白聚糖和II型胶原蛋白细胞外基质。胰岛素样生长因子-I（2 - 10 ng/ml）增加了这两种蛋白质的合成并掺入基质中。TGF-β（2 - 10 ng/ml）也增加了蛋白聚糖的合成。然而，它抑制了蛋白聚糖的沉积，推测是通过增加分子的降解，如聚集蛋白聚糖片段释放到组织培养基中增加所示。TGF-β对II型胶原蛋白沉积没有影响。在牛鼻或关节软骨细胞的微团培养中，20 ng/ml的IL-1α诱导了蛋白聚糖和II型胶原蛋白的显著降解。对胶原蛋白的影响显然涉及其三螺旋的蛋白水解切割，因为基质中解旋的II型胶原蛋白比例增加，以及II型胶原蛋白总量的减少。在完整牛关节软骨的外植体培养中，用50 ng/ml的IL-1α孵育刺激了蛋白聚糖的显著降解，但没有II型胶原蛋白的降解。这些结果表明，尽管关节软骨细胞在分离时能够降解II型胶原蛋白，但它们在原位并不这样做，推测是由于成熟细胞外基质的一些固有特性。本研究证明了微团培养在研究软骨细胞介导的软骨基质周转及其受细胞因子和生长因子调节方面的实用性。
