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

发布时间：2018-05-05 23:30

本文选题：肌腱干细胞 + 循环牵拉　；参考：《中国修复重建外科杂志》2017年01期

【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(P0.05),30 min时出现峰值(P0.05),至60 min时表达量恢复至起始水平(P0.05);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(P0.05)。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(P0.05)。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(P0.05);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(P0.05)。结论循环牵拉能够影响大鼠跟腱来源TSCs立早基因c-fos的m RNA表达,并且呈时间和强度依赖性;提示机械刺激的时间和强度可能在作用早期即对TSCs的分化产生影响,对进一步研究TSCs机械-细胞内信号耦联机制具有重要意义。
[Abstract]:Objective to investigate the effect of mechanical stimulation on c-fos expression of tendon stem cells (TSCs) in rat tendon stem cells derived from Achilles tendon. Methods the Achilles tendon of 8-week-old male SD rats was isolated and cultured by enzyme digestion. The third passage cells were used in the experiment. In this paper, we used the single pull cycle drawing load model at 1 Hz, and used 4% and 8% stretch strength to pull the cells, and collected the cells after pulling 0 ~ (5 ~ 5) ~ 1530 ~ (30) ~ 60120 min for real-time fluorescence quantitative PCR detection, and observed the relative expression of c-fos _ m RNA with the pulling time. And find the highest expression time point Tmax. Then the cells were collected with 8% and 12% tensile strength of 2% and 4%, respectively, and the relative expression of c-fos m RNA was observed. Then, with 8% and 12% tensile strength of 2% and 4%, respectively, the relative expression of c-fos m RNA was observed when the relative expression of c-fos m RNA was stimulated for a short period of time. Finally, the type I collagen, a marker gene of tendon differentiation, was detected with 4% and 8% stretch strength of Tmax1 for 120 min, respectively. The expression of tenomodulinus tenomonas tenomodulinus TNMDN, osteoblast differentiation marker gene Runx2, distal deletion gene 5(distal-less homeobox 5, fatty acid binding protein 4(fatty acid binding protein 4 and FABP4) were detected. Results compared with 0 min, the relative expression of c-fos m RNA at 4% and 8% tensile strength was significantly increased at 15 min, the peak value of P0.05 was found at 30 min, and returned to the initial level at 60 min, so Tmax was 30 min. At 30 min, the relative expression of c-fos m RNA was increased by 2% and 6% and 12% respectively, and the tensile strength of 6% and 12% was higher than that of 2% and 4%, the difference was statistically significant (P 0.05). The relative expression of c-fos m RNA was increased at 30 min and 120 min after a short stretch of 5 min, and the relative expression of c-fos m RNA was increased at 30 min and 120 min. There was no significant difference in the relative expression of all differentiation marker genes between 0 min and 0 min, but the relative expression of Runx2 gene increased significantly at 30 min of traction and 120 min after pulling at 8% retraction intensity. The relative expression of Dlx5, Runx2 and other genes in type I collagen was increased, and the difference was statistically significant (P 0.05). Conclusion cyclic retraction can affect the expression of m RNA in c-fos of TSCs gene derived from Achilles tendon in a time-and intensity-dependent manner, suggesting that the time and intensity of mechanical stimulation may have an effect on the differentiation of TSCs in the early stage of action. It is of great significance to further study the mechanism of mechanical-cellular signal coupling in TSCs.
【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项目(81230040)~~
【分类号】：R686

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