Gao Shang, Tang Kanglai, Zhang Jiqiang, Yang Zhijin, Cui Haifeng, Li Pao, Tang Hong, Zhou Mei
Department of Orthopedics, Orthopedic Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P.R.China.
Department of Orthopedics, Orthopedic Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, 400038,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Jan 15;31(1):85-90. doi: 10.7507/1002-1892.201609014.
To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs) .
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 1 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 T by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T 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 T 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)].
Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes ( <0.05), reached the maximum at 30 minutes ( <0.05), and returned to baseline at 60 minutes ( >0.05) when compared with expression at 0 minute. Therefore, T 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% ( <0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes ( <0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity ( >0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity ( <0.05).
Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cells , and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.
探讨机械拉伸刺激是否影响大鼠跟腱来源的肌腱干细胞（TSCs）中即早基因c-fos mRNA的表达。
采用酶消化法从8周龄雄性Sprague Dawley大鼠的跟腱中分离TSCs，并培养3代。将TSCs分别在1 Hz条件下，用单轴循环拉伸加载系统以4%或8%的拉伸强度刺激0、5、15、30、60和120分钟。在每个时间点，收集TSCs，通过实时荧光定量PCR检测c-fos mRNA表达，找出最佳时间点T。然后，用2%、4%、6%、8%或12%的拉伸强度对TSCs进行T时间的模拟，观察不同拉伸强度下c-fos mRNA的相对表达。接下来，分别对TSCs进行0、5或15分钟的拉伸，然后在松弛状态下孵育至T时间，观察短期刺激后c-fos mRNA表达的变化。最后，分别用4%或8%的拉伸强度对TSCs刺激0、T或120分钟，检测成腱分化相关基因[Ⅰ型胶原、肌腱调节蛋白（TNMD）]、成骨分化相关基因[ runt相关转录因子2（Runx2）、远端缺失同源盒5（Dlx5）]和脂肪生成分化相关基因[脂肪酸结合蛋白4（FABP4）]的表达。
与0分钟时的表达相比，在4%或8%的拉伸强度下，c-fos mRNA的相对表达在15分钟时显著增加（P<0.05），在30分钟时达到最大值（P<0.05），并在60分钟时恢复到基线水平（P>0.05）。因此，T为30分钟。2%的拉伸强度足以在30分钟时引起c-fos mRNA的表达，且在6%、8%和12%的拉伸强度下的表达显著高于2%和4%（P<0.05）。即使是5分钟的短期刺激，c-fos mRNA表达在30分钟时仍可显著增加（P<0.05）。在4%的拉伸强度下，30分钟和120分钟时分化相关基因的相对表达与0分钟时相比无显著差异（P>0.05）；但在8%的拉伸强度下，Runx2基因的相对表达在30分钟时显著增加，Ⅰ型胶原、TNMD、Dlx5和Runx2的相对表达在120分钟时增加（P<0.05）。
机械拉伸刺激可影响大鼠跟腱来源的肌腱干细胞中即早基因c-fos mRNA的相对表达，且具有时间和强度依赖性。提示不同时间或强度的机械刺激可能在早期影响TSCs的分化。本研究对进一步研究TSCs细胞内机械信号转导机制具有重要意义。
