不同机械牵伸条件对大鼠肌腱干细胞分化的影响  

Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells

作　　者：李跑[1] 高尚[1] 周梅[1] 唐红[1] 穆米多 张吉强[2] 唐康来[1] 

机构地区：[1]第三军医大学西南医院骨科全军矫形外科中心,重庆400038 [2]第三军医大学神经生物教研室,重庆400038

出　　处：《中国修复重建外科杂志》2017年第4期481-488,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金重点资助项目(81230040);全军医学科研基金创新专项重点资助项目(13CXZ008)~~

摘　　要：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�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 compa

关 键 词：机械牵拉 肌腱干细胞 分化 大鼠 

分 类 号：R686.1[医药卫生—骨科学]