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目的探讨不同机械牵伸条件对大鼠肌腱干细胞(tendon stem cells,TSCs)RhoA/Rho相关蛋白激酶(Rho associated protein kinases,ROCK)分子表达的影响。方法取2~3月龄雄性SD大鼠(体重200~250 g)跟腱组织,采用酶消化法分离培养TSCs。取第2~3代细胞接种于微沟槽培养皿中,分别以牵伸应变量4%、8%(4%牵伸组、8%牵伸组),牵伸频率1 Hz,牵伸时间4 h/d进行机械牵伸,倒置相差显微镜下观察细胞生长情况;牵伸1、2、3 d后收集细胞,采用实时定量PCR、Western blot检测RhoA、ROCK基因及蛋白表达情况,应用细胞计数试剂盒8检测牵伸后细胞增殖情况。以未牵伸的TSCs作为对照组。结果第2代TSCs形态呈鹅卵石样,聚集生长;接种于微沟槽培养皿后呈杂乱生长;牵伸后细胞贴壁良好,无脱落,并沿受力方向排列。各时间点TSCs中RhoA、ROCK基因相对表达量随牵伸应变量的增加呈递增趋势,组间比较差异均有统计学意义(P<0.05)。牵伸1 d时,4%、8%牵伸组与对照组相比RhoA、ROCK蛋白表达水平相似(P>0.05);2、3 d时,4%、8%牵伸组表达水平显著高于对照组,且随牵伸应变量增加呈递增趋势,组间比较差异均有统计学意义(P<0.05);各时间点,8%牵伸组细胞增殖显著低于4%牵伸组及对照组(P<0.05);4%牵伸组与对照组比较,差异无统计学意义(P>0.05)。结论大鼠TSCs中RhoA、ROCK分子表达水平随牵伸应变量的增加而增加,RhoA/ROCK分子可能是TSCs机械牵伸后的重要效应分子。
Objective To investigate the effect of different mechanical drafting conditions on the RhoA / Rho-associated protein kinase (ROCK) expression in rat tendon stem cells (TSCs). Methods Twenty-three-month-old male Sprague-Dawley rats weighing 200-250 g were randomly divided into three groups: Achilles tendon tissue. TSCs were isolated and cultured by enzyme digestion. The second to third passage cells were inoculated into the microgrooves and cultured in the medium of 4%, 4%, 4% and 8% draft respectively, the draft frequency of 1 Hz, the drawing time 4 h / d mechanical drawing, inverted phase contrast microscope to observe the cell growth; drafting 1, 2, 3 d after the collection of cells, using real-time quantitative PCR, Western blot detection RhoA, ROCK gene and protein expression, Kit 8 detects cell proliferation after drafting. Undrawn TSCs were used as control. Results The second generation of TSCs showed pebble-like morphology and aggregated growth. After inoculation into microgrooves, the TSCs grew in a disorderly manner. After stretching, the cells adhered well and did not fall off and arranged along the stress direction. The relative expression of RhoA and ROCK in TSCs at each time point showed an increasing trend with the increasing amount of distraction strain, with significant difference between the two groups (P <0.05). Compared with the control group, the RhoA and ROCK protein expression levels in the 4% and 8% draft groups were similar (P> 0.05) at 1 d after drafting, and at 4 and 8% drafting groups at 2 and 3 d (P <0.05). At each time point, the cell proliferation in the 8% stretch group was significantly lower than that in the 4% stretch group and the difference between the two groups was statistically significant Control group (P <0.05); 4% drafting group and control group, the difference was not statistically significant (P> 0.05). Conclusion The expression of RhoA and ROCK in rat TSCs increased with the increase of the amount of distraction. RhoA / ROCK may be an important effector of TSCs after mechanical drawing.