OBJECTIVE: To isolate the tendon stem cells (TSCs) from rat patellar tendon and to investigate the effect of mechanical stretching on the expression of Sox-9. METHODS: TSCs were isolated from Sprague Dawley rat (12 weeks old) patellar tendon by collagenase digestion and low density culture. The cell colony morphology and number were observed by crystal violet staining; the cell morphology was observed by inverted phase contrast microscope, and the immunophenotypes of mesenchymal stem cells (MSCs) were determined by flow cytometry. The TSCs at passage 3 was given the mechanical stretching at 4%, 0.17 Hz for 4 hours and 24 hours in the experimental group, and cells without stretching was used as control. The Sox-9 gene and protein expressions were detected by real-time fluorescence quantitative PCR and Western blot. RESULTS: Primary cells showed clonal growth and star shape; after subculture, cells at passage 1 showed fibroblast-like shape. The cells formed cell colonies after 7 days; the expressions were positive for CD29, CD44, and CD90 and negative for CD45. The result of real-time fluorescence quantitative PCR showed that Sox-9 gene was down-regulated at 4 hours after mechanical stretching compared with control (P < 0.05), and up-regulated at 24 hours after mechanical stretching when compared with control group (P < 0.05). The result of Western blot showed that Sox-9 protein expression was lower at 4 hours after stretching, but higher at 24 hours after mechanical stretching than that in control group (P < 0.05). CONCLUSION: The rat patellar TSCs can be isolated successfully, and mechanical stretching inhibits the Sox-9 expression, but the inhibited effect might stimulate the Sox-9 expression after the mechanical stretching effect disappears.
OBJECTIVE: To isolate the tendon stem cells (TSCs) from rat patellar tendon and to investigate the effect of mechanical stretching on the expression of Sox-9. METHODS: TSCs were isolated from Sprague Dawley rat (12 weeks old) patellar tendon by collagenase digestion and low density culture. The cell colony morphology and number were observed by crystal violet staining; the cell morphology was observed by inverted phase contrast microscope, and the immunophenotypes of mesenchymal stem cells (MSCs) were determined by flow cytometry. The TSCs at passage 3 was given the mechanical stretching at 4%, 0.17 Hz for 4 hours and 24 hours in the experimental group, and cells without stretching was used as control. The Sox-9 gene and protein expressions were detected by real-time fluorescence quantitative PCR and Western blot. RESULTS: Primary cells showed clonal growth and star shape; after subculture, cells at passage 1 showed fibroblast-like shape. The cells formed cell colonies after 7 days; the expressions were positive for CD29, CD44, and CD90 and negative for CD45. The result of real-time fluorescence quantitative PCR showed that Sox-9 gene was down-regulated at 4 hours after mechanical stretching compared with control (P < 0.05), and up-regulated at 24 hours after mechanical stretching when compared with control group (P < 0.05). The result of Western blot showed that Sox-9 protein expression was lower at 4 hours after stretching, but higher at 24 hours after mechanical stretching than that in control group (P < 0.05). CONCLUSION: The rat patellar TSCs can be isolated successfully, and mechanical stretching inhibits the Sox-9 expression, but the inhibited effect might stimulate the Sox-9 expression after the mechanical stretching effect disappears.