不同机械牵伸条件对大鼠肌腱干细胞分化的影响

发布时间：2018-03-27 15:04

本文选题：机械牵拉　切入点：肌腱干细胞　出处：《中国修复重建外科杂志》2017年04期

【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(P0.05);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(P0.05);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(P0.05)。Western blot检测示:B组牵拉各时间点TNC蛋白表达均高于E组(P0.05),同时牵拉24 h与E组相比CEBPα表达有显著抑制作用(P0.05);C组牵拉24 h RUNX2蛋白表达显著高于E组(P0.05),同时牵拉24、48 h TNC蛋白表达显著低于E组(P0.05);D组牵拉48 h CEBPα蛋白表达显著高于E组(P0.05),TNC蛋白表达显著低于E组(P0.05),RUNX2蛋白表达与E组比较差异无统计学意义(P0.05)。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。
[Abstract]:Objective to investigate the effects of different mechanical drafting conditions on the differentiation of tendon stem cells (TSCs) from tendon stem cells, and to find out the best uniaxial cyclic drafting load for TSCs tendon differentiation, osteogenic differentiation and adipogenic differentiation. Methods the Achilles tendon of 8-week-old male Sprague-Dawley rats was obtained. TSCs were isolated and cultured by enzyme digestion. TSCs of the third generation were randomly divided into two groups: group A (group A) and group A (group A) and group E (control group). The pulling strength of group 1 was 8 and that of group D was 8 and that of group D was 2 Hz.Using the uniaxial retraction equipment of extracorporeal cells developed by the research group, The cells in group E were cultured statically along the long axis of culture dish for uniaxial circulatory mechanical drafting, and the cells in each group were collected after 48 hours of treatment. Real-time fluorescence quantitative PCR was used to detect the genes associated with tendon differentiation, such as Scleraxis Scleraxis, C(Tenascin, CCAAT/ enhancer, CCAAT-enhancer-binding protein 伪, Lipoprteinlipase LPL2, and RUNX2, which are associated with osteogenic differentiation, and the CCAAT/ enhancer of adipogenic differentiation related gene, CCAAT-enhancer-binding protein 伪, Lipoprteinlipase LPL2, and RUNX2, which are associated with osteogenic differentiation, are detected by real-time fluorescence quantitative PCR. The expression of 5(distal-less homeobox 5 (DLX5) was detected by Western blot. Results Real-time quantitative PCR analysis showed that the relative expression of RNA in group B was significantly higher than that in other groups at 24 h. The relative expression of CEBP 伪 -LPLM RNA in group D was significantly higher than that in other groups at 48 h, and the relative expression of RUNX2 + DLX5 m RNA in group C was significantly higher than that in other groups at 24 h. The expression of TNC protein in group B was higher than that in group E at all time points, and the expression of CEBP 伪 was significantly inhibited at 24 h compared with that in group E. The expression of RUNX2 protein in group C was significantly higher than that in group E at 24 h. At the same time, the expression of TNC protein in group E was significantly lower than that in group E at 48 h. The expression of CEBP 伪 protein in group D was significantly higher than that in group E (P 0.05). There was no significant difference in the expression of RUNX2 protein between group E and group E. Conclusion Mechanical force has no significant difference between group E and group E. Conclusion the expression of CEBP 伪 protein in group E is significantly lower than that in group E (P 0.05). Conclusion the expression of RUNX2 protein in group E is significantly lower than that in group E (P 0.05). Stimuli can promote the differentiation of TSCs. Moreover, the optimal condition of tendon differentiation was that the pulling load of different conditions would cause differentiation in different directions. 4Hz for 24 h was the best condition for tendon differentiation. The best condition for osteogenic differentiation was the best condition for osteogenic differentiation, the best condition for osteogenic differentiation was the best condition for osteogenic differentiation, and the best condition for adipogenic differentiation was 82Hz retraction for 48 h.
【作者单位】：第三军医大学西南医院骨科全军矫形外科中心;第三军医大学神经生物教研室;
【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)~~
【分类号】：R686.1

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