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 m RNA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 m RNA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 m RNA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。 Objective To investigate the effect of different mechanical drafting conditions on the differentiation of tendon stem cells (TSCs), and to find out the best uniaxial cyclic draft load of differentiation, osteogenic differentiation and adipogenic differentiation of TSCs. Methods Achilles tendon was obtained from 8-week-old male Sprague-Dawley rats. TSCs were isolated and cultured by enzyme digestion. The third generation of TSCs were randomly divided into four groups: experimental group A ~ D group and static culture group (control group E), in which group A had a tensile strength of 4%, a frequency of 1 Hz, 4% strength, 2 Hz frequency, 8% tensile strength in Group C, 1 Hz frequency, 8% tensile strength in Group D, and 2 Hz frequency. In vitro cell uniaxial stretching equipment developed by the research group, the cells in groups A ~ D were mechanically drafted along the long axis of the dish, and the cells in group E were cultured in static state. The cells of each group were collected for 12, 24 and 48 hours after treatment, and the expression of Scleraxis (SCX), Tenascin C (TNC), and adipogenic differentiation related genes CCAAT / CCAAT-enhancer-binding protein-α (CEBPα), lipoprtein lipase (LPL) and osteogenic differentiation-related gene RUNX2, distal-less homeobox 5 Expression of TNC, CEBPα and RUNX2 protein were detected by Western blot. Results The results of real-time PCR showed that the relative expression of SCX and TNC m RNA in B group was significantly higher than that in other groups (P <0.05) at 24 h, and the relative expression of CEBPα and LPL m RNA (P <0.05). The relative expression of RUNX2 and DLX5 m RNA in group C was significantly higher than those in other groups after being pulled for 24 h, and the difference was statistically significant There was statistical significance (P <0.05). The results of Western blot showed that the expression of TNC protein in group B was significantly higher than that in group E (P <0.05), while the expression of CEBPα was significantly inhibited in group B at 24 h compared with group E (P <0.05) The expression of RUNX2 protein at 24 h after stretch was significantly higher than that at E (P <0.05), and the protein expression of TNC at 24 h and 48 h after stretch was significantly lower than that at E (P <0.05). The protein expression of CEBPα at 48 h after stretch in D group was significantly higher In group E (P <0.05), TNC protein expression was significantly lower than that in E group (P <0.05). There was no significant difference in RUNX2 protein expression between E group and E group (P> 0.05). Conclusion The mechanical stimulation can promote the differentiation of TSCs, and the traction load of different conditions can cause differentiation in different directions. The optimal conditions for osteogenic differentiation were 4%, 2 Hz, 24 h, 8%, 1 Hz, 24 h, respectively. .