Qin Shengnan, Wang Wen, Fu Shiquan, Cheng Yushan, Chen Honghui, Dong Fei, Chen Qiming, Li Aiguo
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2015 Jul;29(7):884-8.
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.
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.
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).
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.
从大鼠髌腱中分离肌腱干细胞（TSCs），并研究机械拉伸对Sox-9表达的影响。
采用胶原酶消化和低密度培养法从12周龄的Sprague Dawley大鼠髌腱中分离TSCs。通过结晶紫染色观察细胞集落形态和数量；用倒置相差显微镜观察细胞形态，采用流式细胞术检测间充质干细胞（MSCs）的免疫表型。实验组对第3代TSCs给予4%、0.17 Hz的机械拉伸4小时和24小时，未拉伸的细胞作为对照。采用实时荧光定量PCR和蛋白质印迹法检测Sox-9基因和蛋白表达。
原代细胞呈克隆性生长且呈星形；传代后，第1代细胞呈成纤维细胞样形态。细胞在7天后形成细胞集落；CD29、CD44和CD90表达阳性，CD45表达阴性。实时荧光定量PCR结果显示，与对照组相比，机械拉伸4小时后Sox-9基因表达下调（P < 0.05），与对照组相比，机械拉伸24小时后上调（P < 0.05）。蛋白质印迹结果显示，拉伸后4小时Sox-9蛋白表达较低，但机械拉伸24小时后高于对照组（P < 0.05）。
大鼠髌腱TSCs可成功分离，机械拉伸抑制Sox-9表达，但这种抑制作用在机械拉伸作用消失后可能会刺激Sox-9表达。
