Frazer A, Bunning R A, Thavarajah M, Seid J M, Russell R G
Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, UK.
Osteoarthritis Cartilage. 1994 Dec;2(4):235-45. doi: 10.1016/s1063-4584(05)80075-5.
Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.
II型胶原蛋白和聚集蛋白聚糖是关节软骨细胞外基质的主要成分。它们的生物合成和分解代谢受软骨细胞调控。它们可作为体外培养细胞软骨细胞表型的标志物。通过聚合酶链反应（PCR）扩增II型胶原蛋白特异性序列来检测II型胶原蛋白基因表达，该序列使用从新鲜分离和培养的人关节软骨细胞中提取的mRNA经逆转录产生的cDNA。通过在软骨切片和培养的软骨细胞中对II型胶原蛋白进行免疫组织化学定位来确认基因产物的合成。聚集蛋白聚糖核心蛋白也在软骨切片和培养的软骨细胞中进行免疫定位。在皮肤或骨骼中未通过免疫组织化学检测到II型胶原蛋白或聚集蛋白聚糖的表达。这些结果表明，通过联合应用PCR和免疫组织化学，可以在培养中对人关节软骨细胞进行表征。白细胞介素-1β（IL-1β）可能在关节炎中软骨基质的破坏中起重要作用，而转化生长因子-β（TGFβ）可能具有相反的作用，它们的联合作用可能调节软骨细胞表型。研究了重组人IL-1β（rhIL-1β）和重组人TGFβ（rhTGFβ）对培养的软骨细胞产生II型胶原蛋白的影响。结果表明，TGFβ增强了培养的软骨细胞中通过免疫细胞化学定位的II型胶原蛋白的产生。IL-1β抑制II型胶原蛋白mRNA的表达。讨论了这项研究对于更好地理解退行性软骨疾病的意义。
