Alfredson Håkan, Lorentzon Mattias, Bäckman Stina, Bäckman Assar, Lerner Ulf H
Department of Surgical and Perioperative Science, Sports Medicine Unit, University of Umeå, S-901 87 Umeå, Sweden.
J Orthop Res. 2003 Nov;21(6):970-5. doi: 10.1016/S0736-0266(03)00107-4.
The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1+/-4.3 (years+/-SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at -80 degrees C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled (32P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrix-metalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue compared with control tissue. Using real-time PCR, 4/5 and 3/5 patients showed up-regulation of MMP-2 and FNRB mRNA, respectively. For decorin, VEGF, and MAPKp38, real-time PCR revealed a great variability among patients. Interestingly, the mRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue.
慢性疼痛性跟腱病的病因和发病机制尚不清楚。本研究旨在使用cDNA阵列和实时定量聚合酶链反应（实时PCR）技术研究跟腱病组织和对照组织样本。纳入了5例慢性疼痛性跟腱病患者（女性，平均年龄57.1±4.3岁（岁±标准差））。从所有患者身上，在跟腱病区域取一份活检组织，在肌腱临床正常区域（对照）取一份活检组织。组织样本立即浸入RNA保护剂中，并在-80℃下冷冻，直至提取RNA。分别从对照部位和跟腱病部位提取的部分合并RNA被转录为cDNA，进行放射性标记（32P），与cDNA表达阵列杂交，并在磷光成像屏上曝光过夜。使用实时PCR分析在cDNA阵列分析中显示受调控的特定基因在个体样本中的表达。cDNA阵列显示，与对照组织相比，跟腱病组织中基质金属蛋白酶-2（MMP-2）、纤连蛋白亚基B（FNRB）、血管内皮生长因子（VEGF）和丝裂原活化蛋白激酶p38（MAPKp38）的基因表达上调，而基质金属蛋白酶-3（MMP-3）和核心蛋白聚糖下调。使用实时PCR，4/5和3/5的患者分别显示MMP-2和FNRB mRNA上调。对于核心蛋白聚糖、VEGF和MAPKp38，实时PCR显示患者之间存在很大差异。有趣的是，几种细胞因子和细胞因子受体的mRNA未受调控，表明慢性疼痛性跟腱病不存在炎症过程。总之，cDNA阵列和实时PCR可用于研究跟腱病组织和对照肌腱组织之间基因表达水平的差异。
