Objective: To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation.
Methods: TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot.
Results: The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P>0.05).
Conclusion: The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.
目的: 探讨不同机械牵伸条件对肌腱干细胞（tendon stem cells，TSCs）分化的影响，寻求 TSCs 成肌腱分化、成骨分化以及成脂肪分化的最佳单轴循环牵伸载荷。.
方法: 取 8 周龄雄性 SD 大鼠跟腱，采用酶消化法分离培养 TSCs。取第 3 代 TSCs，随机分为不同牵拉条件组（实验组 A～D 组）及静态培养组（对照组 E 组），其中 A 组牵拉强度 4%、频率 1 Hz，B 组牵拉强度 4%、频率 2 Hz，C 组牵拉强度 8%、频率 1 Hz，D 组牵拉强度 8%、频率 2 Hz。利用课题组自行研发的体外细胞单轴循环牵拉设备，沿培养皿长轴对 A～D 组细胞进行单轴循环机械牵伸，E 组细胞行静态培养。分别处理 12、24、48 h 后收集各组细胞，采用实时荧光定量 PCR 检测成腱分化相关基因 Scleraxis（SCX）、抗细胞黏合素 C（Tenascin C，TNC），成脂肪分化相关基因 CCAAT/增强子结合蛋白-α（CCAAT-enhancer-binding protein-α，CEBPα）、脂蛋白脂肪酶（lipoprteinlipase，LPL）及成骨分化相关基因 RUNX2、远端缺失基因 5（distal-less homeobox 5，DLX5）的表达；Western blot 检测 TNC、CEBPα 及 RUNX2 蛋白表达。.
结果: 实时荧光定量 PCR 检测示：SCX、TNC mRNA 相对表达量在 B 组牵拉 24 h 时显著高于其余各组，差异有统计学意义（ P<0.05）；CEBPα、LPL mRNA 相对表达量在 D 组牵拉 48 h 时显著高于其余各组，差异有统计学意义（ P<0.05）；RUNX2、DLX5 mRNA 相对表达量在 C 组牵拉 24 h 时显著高于其余各组，差异有统计学意义（ P<0.05）。Western blot 检测示：B 组牵拉各时间点 TNC 蛋白表达均高于 E 组（ P<0.05），同时牵拉 24 h 与 E 组相比 CEBPα 表达有显著抑制作用（ P<0.05）；C 组牵拉 24 h RUNX2 蛋白表达显著高于 E 组（ P<0.05），同时牵拉 24、48 h TNC 蛋白表达显著低于 E 组（ P<0.05）；D 组牵拉 48 h CEBPα 蛋白表达显著高于 E 组（ P<0.05），TNC 蛋白表达显著低于 E 组（ P<0.05），RUNX2 蛋白表达与 E 组比较差异无统计学意义（ P>0.05）。.
结论: 机械力学刺激可以促进 TSCs 发生分化，而且不同条件的牵拉载荷会引起不同方向分化。4%、2 Hz 牵拉 24 h 为成腱分化最佳条件，8%、1 Hz 牵拉 24 h 为成骨分化最佳条件，8%、2 Hz 牵拉 48 h 为成脂肪分化最佳条件。.
Keywords: Mechanical stretch; differentiation; rat; tendon stem cells.