Martin I, Jakob M, Schäfer D, Dick W, Spagnoli G, Heberer M
Department of Surgery, Research Division, University of Basel, Switzerland.
Osteoarthritis Cartilage. 2001 Feb;9(2):112-8. doi: 10.1053/joca.2000.0366.
To quantify the expression of genes encoding extracellular matrix (ECM) proteins in human cartilage from normal and osteoarthritic (OA) joints.
Human cartilage samples were classified as control (CTR) or OA according to clinical evaluation and assessed histologically and biochemically to confirm the diagnosis. mRNAs encoding collagen types I, II and X, aggrecan, versican, osteopontin and osteocalcin were quantified by real-time reverse transcription-PCR assays and normalized to a reference mRNA (GAPDH).
RNA from native cartilage could be reproducibly and efficiently amplified by real-time PCR only if isolated using purification membranes. Primers and fluorescent probes for real-time PCR, endowed with comparable (<6% difference from GAPDH) and high (>91%) amplification efficiencies, were designed and validated for the selected ECM genes. The expression of most genes under investigation displayed large variations and was not significantly different in CTR and OA cartilage. Only osteopontin mRNA levels were significantly higher in OA than CTR specimens. mRNA ratios of collagen type II to I and of aggrecan to versican, defined as indexes of chondrocyte differentiation, were less variable within each population than the single genes and markedly higher (27.0 and 7.6-fold, respectively) in CTR than OA cartilage, with high statistical significance (P = 0.00013 and P = 0.00007, respectively).
Our results provide evidence that gene patterns related to chondrocyte differentiation discriminate between CTR and OA human cartilage with higher sensitivity than single ECM genes. The method described here has the potential to improve understanding of the progression of OA and could become a valuable diagnostic tool.
量化正常和骨关节炎（OA）关节的人软骨中编码细胞外基质（ECM）蛋白的基因表达。
根据临床评估将人软骨样本分类为对照组（CTR）或OA组，并进行组织学和生化评估以确诊。通过实时逆转录 - PCR测定法对编码I型、II型和X型胶原蛋白、聚集蛋白聚糖、多功能蛋白聚糖、骨桥蛋白和骨钙素的mRNA进行定量，并将其标准化为参考mRNA（甘油醛 - 3 - 磷酸脱氢酶，GAPDH）。
只有使用纯化膜分离时，天然软骨的RNA才能通过实时PCR进行可重复且高效的扩增。针对所选ECM基因设计并验证了实时PCR的引物和荧光探针，其具有可比的（与GAPDH的差异<6%）和高的（>91%）扩增效率。大多数研究基因的表达表现出很大差异，在CTR和OA软骨中无显著差异。仅骨桥蛋白mRNA水平在OA标本中显著高于CTR标本。定义为软骨细胞分化指标的II型与I型胶原蛋白的mRNA比率以及聚集蛋白聚糖与多功能蛋白聚糖的mRNA比率，在每个群体中比单个基因的变异性小，并且在CTR软骨中比OA软骨显著更高（分别为27.0倍和7.6倍），具有高度统计学意义（分别为P = 0.00013和P = 0.00007）。
我们的结果提供了证据，表明与软骨细胞分化相关的基因模式区分CTR和OA人软骨的敏感性高于单个ECM基因。本文所述方法有可能增进对OA进展的理解，并可能成为一种有价值的诊断工具。
