cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic Achilles tendinosis.
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.
Alfredson
Håkan
H
Department of Surgical and Perioperative Science, Sports Medicine Unit, University of Umeå, S-901 87 Umeå, Sweden. hakan.alfredson@idrott.umu.se
Lorentzon
Mattias
M
Bäckman
Stina
S
Bäckman
Assar
A
Lerner
Ulf H
UH
D000125:Achilles Tendon / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D058575:Decorin; D016326:Extracellular Matrix Proteins; D005260:Female; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052256:Tendinopathy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D011509:Proteoglycans; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020782:Matrix Metalloproteinases