循环牵拉对大鼠跟腱来源肌腱干细胞c-fos基因表达的影响  

Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells

作　　者：高尚[1] 唐康来[1] 张吉强[2] 杨志金[1] 崔海峰[1] 李跑[1] 唐红[1] 周梅[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1期85-90,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金重点项目(81230040)~~

摘　　要：目的探讨机械刺激对大鼠跟腱来源肌腱干细胞(tendon stem cells,TSCs)立早基因c-fos表达的影响。方法取8周龄雄性SD大鼠跟腱组织,用酶消化法分离、培养TSCs,取第3代细胞用于实验。利用单拉循环牵伸载荷模型在1 Hz条件下,分别用4%和8%牵拉强度牵拉细胞,并在牵拉0、5、15、30、60、120 min后收集细胞进行实时荧光定量PCR检测,观察c-fos m RNA相对表达量随牵拉时间的变化,并找到表达最高时间点Tmax。然后分别用2%、4%、6%、8%和12%的牵拉强度,在牵拉Tmax时收集细胞,观察c-fos m RNA相对表达量随牵拉强度的变化。接着分别用2%、4%、6%、8%和12%的牵拉强度,对TSCs经0、5、15 min短时间牵拉后静置至Tmax,观察c-fos m RNA相对表达量经短时刺激后的变化情况。最后分别用4%和8%牵拉强度牵拉细胞0、Tmax、120 min,检测成肌腱分化标志基因Ⅰ型胶原、腱调蛋白(tenomodulin,TNMD),成骨分化标志基因Runx2、远端缺失基因5(distal-less homeobox 5,Dlx5),成脂肪分化标志基因脂肪酸结合蛋白4(fatty acid binding protein 4,FABP4)的表达情况。结果与0 min相比,在4%和8%牵拉强度下,c-fos m RNA相对表达量在牵拉15 min时显著升高(P<0.05),30 min时出现峰值(P<0.05),至60 min时表达量恢复至起始水平(P>0.05);故Tmax为30 min。牵拉30 min时,2%的牵拉强度即可使c-fos m RNA相对表达量升高,且6%、8%和12%牵拉强度较2%、4%牵拉强度进一步升高,差异均有统计学意义(P<0.05)。经过5 min的短时间牵拉,并静置至30 min时即可使c-fos m RNA相对表达量升高(P<0.05)。4%牵拉强度下,在牵拉30 min和120 min时,各分化标志基因相对表达量与0 min比较差异均无统计学意义(P>0.05);而8%牵拉强度下,在牵拉30 min时,Runx2基因相对表达量显著升高,在牵拉120 min时,Ⅰ型胶原、TNMD、Dlx5、Runx2等基因相对表达量均升高,差异均有统计学意义(P<0.05)。结论循环牵拉能够影响大鼠跟腱来源TSCs立早基Objective To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-los mRNA in rat Achilles-derived tendon stem cells （TSCs） in vitro. Methods TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of i Hz,respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point Tmax by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for Tmax to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to Tmax to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin （TNMD）], the osteogenic differentiation related genes [runt related transcription factor 2 （Runx2）, distal-less homeobox 5 （Dlx5）], and the adipogenic differentiation related gene [fatty acid binding protein 4 （FABP4）]. Results Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes （P〈0.05）, reached the maximum at 30 minutes （P〈0.05）, and returned to baseline at 60 minutes （P〉0.05） when compared with expression at 0 minute. Therefore, Tmax was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% （P〈0.05）. Even for a short period stimulation o

关 键 词：肌腱干细胞 循环牵拉 立早基因 c—fos 大鼠 

分 类 号：R686[医药卫生—骨科学]