机械拉伸诱导人骨髓间充质干细胞向肌腱细胞定向分化及其力信号转导的实验研究
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摘要
机体组织/细胞处于复杂的力学微环境中,且其生长发育受到力学微环境的调控。肌腱/韧带组织是一种典型的传递肌肉收缩所产生的应力的连接性组织。它们在发育进程中,长期受到周期性牵拉产生的张应力,这对它们的形成具有重要的调节作用。然而,肌腱/韧带由于过度负载或者老化引起的损伤在日常生活中很常见,而且其预防和治疗仍是临床医生面临的挑战问题之一。
骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BM-MSCs)是一种具有多向分化潜能的成体干细胞,由于其具有来源丰富、取材简单、快速增殖、易于转染外源基因、以及低免疫抗原反应等优点,成为组织工程、基因治疗以及细胞治疗的研究热点。目前已经有研究报道机械刺激可以诱导BM-MSCs向肌腱细胞方向分化。这为利用组织工程实现肌腱/韧带损伤修复带来了新希望,但是其力信号转导和分子机理还不清楚,并成为后续研究工作的重点。
为了进一步研究机械刺激对BM-MSCs向肌腱细胞方向分化的影响以及分化过程中的相关力信号转导。本文以人骨髓间充质干细胞(human bone marrow-derived mesenchymal stem cells, hMSCs)为研究对象,利用拉伸装置(ST-140)对hMSCs加载一定的机械拉伸,通过实时定量PCR检测肌腱相关标记基因的表达情况,筛选诱导hMSCs向肌腱细胞方向分化的机械拉伸条件;并且利用Y-27632、细胞松弛素D和PF 530228 (PF 228)分别抑制RhoA/ROCK、细胞骨架和FAK(Focal adhesion kinase, FAK),考察RhoA/ROCK、细胞骨架和FAK在机械拉伸诱导hMSCs向肌腱细胞方向分化过程中的作用。
本文得到的主要实验结果如下:
①hMSCs多向分化潜能
多向分化潜能通常是指在体外诱导条件下具有分化为成骨细胞、成脂肪细胞和软骨细胞的能力。我们利用成骨诱导分化培养基、成脂肪诱导分化培养基和成软骨诱导分化培养基诱导hMSCs,以MSCs基础培养基培养作为对照。诱导21天后分别进行茜素红、油红O和阿尔新蓝组织化学染色。染色结果显示成骨诱导分化培养基诱导21天后,诱导组茜素红染色呈阳性而对照组呈阴性;成脂肪诱导分化培养基诱导21天后,诱导组油红O染色呈阳性而对照组呈阴性;成软骨诱导分化培养基诱导21天后,诱导组阿尔新蓝染色呈阳性而对照组呈阴性。这表明:我们的研究对象hMSCs,在体外具有分化为成骨细胞、成脂肪细胞和软骨细胞的多向分化能力。
②机械拉伸诱导hMSCs向肌腱细胞方向分化
对hMSCs加载不同条件的机械拉伸(频率:0.1 Hz、1.0 Hz,应变大小:5%、10%、15% strain,拉伸时间:24 h、48 h),通过实时定量RT-PCR和Western blot技术分别检测肌腱相关标记基因如Collagen type I (Col I)、Collagen type III(Col III)、tenascin-C(TNC)和scleraxis(SCX),和肌腱相关标记蛋白如Col I和TNC的表达。实验结果显示:不同的机械拉伸引起的基因和蛋白的表达变化不尽相同。在本研究范围内,1.0 Hz、10% strain,持续时间分别为24 h和48 h能够促进肌腱相关标记基因和蛋白的表达。加载机械拉伸24 h后,Col I、Col III、TNC和SCX的基因表达分别为相应对照组的1.220±0.057倍、1.31±0.087倍、1.355±0.133倍和1.605±0.244倍;Col I和TNC的蛋白表达分别为1.191±0.042倍和1.47±0.047倍。加载机械拉伸48 h后,Col I、Col III、TNC和SCX的基因表达分别为1.285±0.049倍、1.375±0.152倍、1.64±0.146倍和2.503±0.285倍;Col I和TNC的蛋白表达分别为1.258±0.093倍和2.231±0.278倍。这些实验结果表明1.0 Hz、10% strain,持续时间48 h的机械拉伸能够诱导hMSCs向肌腱细胞方向分化。同时我们观察到机械拉伸诱导hMSCs垂直于拉伸方向排列,而且细胞排列的趋势和拉伸频率、拉伸形变和拉伸时间相关。
③Y-27632、细胞松弛素D和PF 228对机械拉伸诱导的actin超微结构的变化的影响
以Fluoresceinisothiocyanate (FITC)-phalloidin标记细胞骨架F-actin,通过激光共聚焦显微镜观察actin的超微结构。利用Y-27632、细胞松弛素D和PF 228分别抑制RhoA/ROCK、细胞骨架和FAK,考察RhoA/ROCK、细胞骨架和FAK对机械拉伸诱导的hMSCs形态和actin超微结构的变化的影响。激光共聚焦显微镜的实验结果显示:静态培养的细胞随机排列,actin沿着细胞长轴呈随机排列;机械拉伸促进actin的聚合,使actin的密度升高,并诱导actin纤维垂直于拉伸方向排列。Y-27632处理的细胞,actin的聚合受到抑制,只有少数actin分布在细胞边缘;Y-27632处理的细胞加载机械拉伸后,actin呈随机排列,密度没有显著变化,但是actin变得不连续并呈点状,细胞边缘出现很短的actin。细胞松弛素D处理的细胞,actin变短且不连续,并在细胞边缘聚积;细胞松弛素D处理的细胞加载机械拉伸后,actin的不连续和点状程度更加明显,且actin呈随机排列。PF 228处理的细胞,actin并无显著变化;PF 228处理的细胞加载机械拉伸后,actin出现撕裂状,部分actin不再沿着细胞长轴排列,而是垂直于拉伸方向排列,而且拉伸后产生一些细胞碎片。这些结果表明RhoA/ROCK和FAK对机械拉伸诱导的actin的变化至关重要,而且机械拉伸通过诱导actin垂直于拉伸方向排列,最终引起细胞垂直于拉伸方向排列。
④Y-27632、细胞松弛素D和PF 228抑制机械拉伸诱导的FAK 397位点Tyr磷酸化及向肌腱细胞方向分化
Y-27632、细胞松弛素D和PF 228抑制机械拉伸诱导的hMSCs FAK磷酸化。Western blot结果显示Y-27632和细胞松弛素D抑制后,FAK磷酸化下降到对照组水平; PF 228处理抑制机械拉伸诱导的FAK磷酸化,将FAK磷酸化水平下降到20%左右,且磷酸化程度比对照水平更低。这表明RhoA/ROCK和细胞骨架对机械拉伸诱导的FAK磷酸化具有重要的调节作用,而PF 228作为FAK的选择性抑制剂对FAK磷酸化的抑制程度更剧烈。不仅如此,实验结果进一步显示:Y-27632、细胞松弛素D或PF 228处理,显著抑制机械拉伸诱导的肌腱相关标记基因(Col I、Col III、TNC、SCX、EphA4、Eya2和Six1)和蛋白(Col I、Col III和TNC)的表达。相比而言,PF 228的抑制效果更加剧烈。这表明RhoA/ROCK、细胞骨架和FAK调节机械拉伸诱导的hMSCs向肌腱细胞方向分化。在这个分化过程中,RhoA/ROCK、细胞骨架和FAK之间相互影响、相互调节,组成一个力学感应的信号网络。这个信号网络感应胞外力学环境,并将力信号转变为生化信号,并进一步触发向肌腱细胞方向分化进程。抑制这个信号网络中的任何一个成员,将导致机械拉伸诱导hMSCs向肌腱细胞方向分化的失败。综合以上研究表明:hMSCs不仅具有分化为成骨细胞、脂肪细胞和软骨细胞的能力,而且在机械拉伸加载条件(1.0 Hz,10% strain)的条件下,能够向肌腱细胞方向分化。在机械拉伸诱导hMSCs向肌腱细胞方向分化过程中,激活的RhoA/ROCK、完整的细胞骨架和FAK Tyr397位点磷酸化是必要条件,而且RhoA/ROCK、细胞骨架和FAK相互影响并协同调节械拉伸诱导hMSCs向肌腱细胞方向分化。本研究对认识机械拉伸诱导hMSCs定向分化的分子基础以及分化过程中力信号传导具有重要意义,还可为临床上腱/韧带创伤的治疗及其组织工程化提供定量的参考依据。
Tendon/ligament is a kind of connective tissue subjected to complicated mechanical microenvironment which is controlling their growth, development and regeneration. However, it is always injured in the daily life, and the cure of the injuries is a serious challenge for clinician.
Mesenchymal stem cells (MSCs) are able to differentiate into a lot of cell lineages. For their advantages, such as abundant source, fast proliferation, easy to transfer exogenous gene, low immune reaction, they become one of the research focus of tissue engineering, gene therapy or cell therapy. At present, it was reported that MSCs were able to differentiate into tenocytes by mechanical stimuli, and to be used for tendon/ligament repair. It brings new hopes for the appliacation of tissue engineering for tendon/ligament repair. However, the mechanical stretch-induced tenogenic differentiation is not well understood and the mechanotransduction mechanism is still unclear. Therefore, it becomes an important research content of in the furthure.
This study further investigated the effect of mechanical stretch on tenogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), and the mechanotransduction mechanism during the process. To search for an appropriate stretch parameter, cyclic stretch device (ST-140) was used to load mechanical stretch on hMSCs which growth on febronectin-coated silicon chambers. After stretch, tendon-related gene and protein expressions were analyzed by real-time RT-PCR and western blot. After that, to examine the effect of RhoA/ROCK, cytoskeletal organization and FAK (Focal adhesion kinase) during mechanical stretch-induced tenogenic differentiation, Y-27632, ctochalasin D and PF 530228 (PF 228) were used to inhibit RhoA/ROCK, cytoskeletal organization and FAK, respectively.
The main experiments and major results are as follows:
①hMSCs multi-lineages differentaiton potential
To examine the multi-lineages differentaiton potential of hMSCs used in this study, hMSCs were cultured in OS (osteogenic) induction media, AD (adipogenic) induction media or CH (chondrogenic) induction media for 21 d. Alizarin red S, oil red O and acian blue staining were conducted to analyzed the OS induction, AD induction and CH induction, respectively. The staining results suggest that hMSCs could differentiate into osteogenic, adipogenic and chondrogenic.
②Mehcanical stretch-induced tenogenic differentiation of hMSCs
Aimed to search for appropriate stretch parameters, hMSCs were loaded with different mechanical stretch (0.1 Hz, 15% strain, 24 h and 48 h; 0.1 Hz, 10% strain, 24 h and 48 h; 0.1 Hz, 5% strain, 24 h and 48 h; 1.0 Hz, 15% strain, 24 h and 48 h; 1.0 Hz, 10% strain, 24 h and 48 h; 1.0 Hz, 5% strain, 24 h and 48 h), gene expressions of collagen type I (Col I), collagen type III (Col III), tenascin-C (TNC) and scleraxis (SCX), and protein expressions of Col I and TNC, were anaylized by real-time RT-PCR and western blot. The results showed that only the mechanical stretch (1.0 Hz, 10% strain, 24 h and 48 h) can promote the gene expressions of Col I, Col III, TNC and SCX,and the protein expressions of Col I and TNC. It suggests that 1.0 Hz, 10% strain mechanical stretch can induce tenogenic differentiation of hMSCs. Concomitant with this process, hMSCs were induced to realign vertically to the direction of stretch, and the trend of realignment was dependent on the strain, frequence and duration of mechanical stretch.
③Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in morphology of hMSCs
Y-27632, cytochalsin D and PF 228 had important effect on mechanical stretch-induced changes in cytoskeletal dynamics which defines cell shape. The changes in density of actin fibers and vertical alignment of actin fibers to the direction of stretch, are due to the adaptation to mechanical loads which can resist and reduce experienced mechanical force. Inhibition of RhoA/ROCK resulted in lacking actin fibers, failure in trailing-edge retraction and leaving a long tail behind cells, and showed stellate-like morphology. However, in the presence of Y-27632 and mechanical stretch resulted in discontinuous actin fibers and very short actin fibers in the cell edges, and no significant changes in cell shape and favorite alignment with respect to the direction of stretch. Cytochalasin D treatment resulted in disruption of actin fibers organization and circular cell shape. When the cytochalasin D treated cells were exposure to stretch, the actin fibers were further disrupted and the cell shape became more circuler. PF 228 incubation resulted in more circular cell shape and shorter actin fibers. Interestingly, PF 228 incubation with mechanical stretch resulted in stellate-like cells, shorter actin fibers and rough edges, and even resulted in generation of cell debris. In addition, there were also no specific alignments of cells and actin fibers with respect to the direction of stretch. These data suggest that mechanical stretch facilitates actin fibers formation and induces realignment of cells by inducing actin fibers perpendicular aligning to the direction of stretch which can resist and adapt to the force balance. And the stretch-induced realignment is needed activation of RhoA/ROCK, intact cytoskeltal organization and FAK phosphorylation.
④Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in FAK phosphorylation and tenogenic differentiation of hMSCs
Western blot showed that with experience of stretch for 30 min, phosphorylation of FAK at Tyr397 was increased up to 1.966±0.167 times. When cells were treated by Y-27632, phosphorylation of FAK at Tyr397 was decreased to 1.081±0.1 times. When the assembling of F-actin was blocked by cytochalasin, phosphorylation of FAK at Tyr397 was decreased to 1.095±0.099 times. Treatment with PF 228 resulted in dramatical decrease of phosphorylation of FAK at Tyr397. It was reduced to 0.395±0.075 times. It indicates RhoA/ROCK and cytoskeletal organization are essential to mechanical stretch-activated FAK phosphorylation.
Except for the influence on morphology and stretch-activated FAK phosphorylation, Y-27632, cytochalsin D and PF 228 have important effect on gene expressions and even to differentiation. Y-27632, cytochalsin D or PF 228 inhibited the mechanical stretch-induced gene expressions of Col I, Col III, TNC, SCX, EphA4, Eya2 and Six1, and the protein expressions of Col I, Col III and TNC. It suggests that RhoA/ROCK, cytoskeleton and FAK are necessary for mechanical stretch-induced tenogenic diferentiation of hMSCs. Moreover, RhoA/ROCK, cytoskeletal organization and FAK interacted with each other in the stretch-induced tenogenic differentiation of hMSCs.
In summary, we can infer that RhoA/ROCK, cytoskeletal dynamics and FAK interacte with each other and compose a signaling network to sense mechanical environment. The signaling network converts mechanical stretch into biochemical signaling, and then triggers stretch-induced tenogenic differentiation. Inhibiting any component of the signaling network, it fails to convert mechanical stretching into biochemical signaling and the stretch-induced tenogenic differentiation will be aborted. It provides novel view into the mechanisms of stretch-induced tenogenesis and supports the therapeutic potential of hMSCs.
骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BM-MSCs)是一种具有多向分化潜能的成体干细胞,由于其具有来源丰富、取材简单、快速增殖、易于转染外源基因、以及低免疫抗原反应等优点,成为组织工程、基因治疗以及细胞治疗的研究热点。目前已经有研究报道机械刺激可以诱导BM-MSCs向肌腱细胞方向分化。这为利用组织工程实现肌腱/韧带损伤修复带来了新希望,但是其力信号转导和分子机理还不清楚,并成为后续研究工作的重点。
为了进一步研究机械刺激对BM-MSCs向肌腱细胞方向分化的影响以及分化过程中的相关力信号转导。本文以人骨髓间充质干细胞(human bone marrow-derived mesenchymal stem cells, hMSCs)为研究对象,利用拉伸装置(ST-140)对hMSCs加载一定的机械拉伸,通过实时定量PCR检测肌腱相关标记基因的表达情况,筛选诱导hMSCs向肌腱细胞方向分化的机械拉伸条件;并且利用Y-27632、细胞松弛素D和PF 530228 (PF 228)分别抑制RhoA/ROCK、细胞骨架和FAK(Focal adhesion kinase, FAK),考察RhoA/ROCK、细胞骨架和FAK在机械拉伸诱导hMSCs向肌腱细胞方向分化过程中的作用。
本文得到的主要实验结果如下:
①hMSCs多向分化潜能
多向分化潜能通常是指在体外诱导条件下具有分化为成骨细胞、成脂肪细胞和软骨细胞的能力。我们利用成骨诱导分化培养基、成脂肪诱导分化培养基和成软骨诱导分化培养基诱导hMSCs,以MSCs基础培养基培养作为对照。诱导21天后分别进行茜素红、油红O和阿尔新蓝组织化学染色。染色结果显示成骨诱导分化培养基诱导21天后,诱导组茜素红染色呈阳性而对照组呈阴性;成脂肪诱导分化培养基诱导21天后,诱导组油红O染色呈阳性而对照组呈阴性;成软骨诱导分化培养基诱导21天后,诱导组阿尔新蓝染色呈阳性而对照组呈阴性。这表明:我们的研究对象hMSCs,在体外具有分化为成骨细胞、成脂肪细胞和软骨细胞的多向分化能力。
②机械拉伸诱导hMSCs向肌腱细胞方向分化
对hMSCs加载不同条件的机械拉伸(频率:0.1 Hz、1.0 Hz,应变大小:5%、10%、15% strain,拉伸时间:24 h、48 h),通过实时定量RT-PCR和Western blot技术分别检测肌腱相关标记基因如Collagen type I (Col I)、Collagen type III(Col III)、tenascin-C(TNC)和scleraxis(SCX),和肌腱相关标记蛋白如Col I和TNC的表达。实验结果显示:不同的机械拉伸引起的基因和蛋白的表达变化不尽相同。在本研究范围内,1.0 Hz、10% strain,持续时间分别为24 h和48 h能够促进肌腱相关标记基因和蛋白的表达。加载机械拉伸24 h后,Col I、Col III、TNC和SCX的基因表达分别为相应对照组的1.220±0.057倍、1.31±0.087倍、1.355±0.133倍和1.605±0.244倍;Col I和TNC的蛋白表达分别为1.191±0.042倍和1.47±0.047倍。加载机械拉伸48 h后,Col I、Col III、TNC和SCX的基因表达分别为1.285±0.049倍、1.375±0.152倍、1.64±0.146倍和2.503±0.285倍;Col I和TNC的蛋白表达分别为1.258±0.093倍和2.231±0.278倍。这些实验结果表明1.0 Hz、10% strain,持续时间48 h的机械拉伸能够诱导hMSCs向肌腱细胞方向分化。同时我们观察到机械拉伸诱导hMSCs垂直于拉伸方向排列,而且细胞排列的趋势和拉伸频率、拉伸形变和拉伸时间相关。
③Y-27632、细胞松弛素D和PF 228对机械拉伸诱导的actin超微结构的变化的影响
以Fluoresceinisothiocyanate (FITC)-phalloidin标记细胞骨架F-actin,通过激光共聚焦显微镜观察actin的超微结构。利用Y-27632、细胞松弛素D和PF 228分别抑制RhoA/ROCK、细胞骨架和FAK,考察RhoA/ROCK、细胞骨架和FAK对机械拉伸诱导的hMSCs形态和actin超微结构的变化的影响。激光共聚焦显微镜的实验结果显示:静态培养的细胞随机排列,actin沿着细胞长轴呈随机排列;机械拉伸促进actin的聚合,使actin的密度升高,并诱导actin纤维垂直于拉伸方向排列。Y-27632处理的细胞,actin的聚合受到抑制,只有少数actin分布在细胞边缘;Y-27632处理的细胞加载机械拉伸后,actin呈随机排列,密度没有显著变化,但是actin变得不连续并呈点状,细胞边缘出现很短的actin。细胞松弛素D处理的细胞,actin变短且不连续,并在细胞边缘聚积;细胞松弛素D处理的细胞加载机械拉伸后,actin的不连续和点状程度更加明显,且actin呈随机排列。PF 228处理的细胞,actin并无显著变化;PF 228处理的细胞加载机械拉伸后,actin出现撕裂状,部分actin不再沿着细胞长轴排列,而是垂直于拉伸方向排列,而且拉伸后产生一些细胞碎片。这些结果表明RhoA/ROCK和FAK对机械拉伸诱导的actin的变化至关重要,而且机械拉伸通过诱导actin垂直于拉伸方向排列,最终引起细胞垂直于拉伸方向排列。
④Y-27632、细胞松弛素D和PF 228抑制机械拉伸诱导的FAK 397位点Tyr磷酸化及向肌腱细胞方向分化
Y-27632、细胞松弛素D和PF 228抑制机械拉伸诱导的hMSCs FAK磷酸化。Western blot结果显示Y-27632和细胞松弛素D抑制后,FAK磷酸化下降到对照组水平; PF 228处理抑制机械拉伸诱导的FAK磷酸化,将FAK磷酸化水平下降到20%左右,且磷酸化程度比对照水平更低。这表明RhoA/ROCK和细胞骨架对机械拉伸诱导的FAK磷酸化具有重要的调节作用,而PF 228作为FAK的选择性抑制剂对FAK磷酸化的抑制程度更剧烈。不仅如此,实验结果进一步显示:Y-27632、细胞松弛素D或PF 228处理,显著抑制机械拉伸诱导的肌腱相关标记基因(Col I、Col III、TNC、SCX、EphA4、Eya2和Six1)和蛋白(Col I、Col III和TNC)的表达。相比而言,PF 228的抑制效果更加剧烈。这表明RhoA/ROCK、细胞骨架和FAK调节机械拉伸诱导的hMSCs向肌腱细胞方向分化。在这个分化过程中,RhoA/ROCK、细胞骨架和FAK之间相互影响、相互调节,组成一个力学感应的信号网络。这个信号网络感应胞外力学环境,并将力信号转变为生化信号,并进一步触发向肌腱细胞方向分化进程。抑制这个信号网络中的任何一个成员,将导致机械拉伸诱导hMSCs向肌腱细胞方向分化的失败。综合以上研究表明:hMSCs不仅具有分化为成骨细胞、脂肪细胞和软骨细胞的能力,而且在机械拉伸加载条件(1.0 Hz,10% strain)的条件下,能够向肌腱细胞方向分化。在机械拉伸诱导hMSCs向肌腱细胞方向分化过程中,激活的RhoA/ROCK、完整的细胞骨架和FAK Tyr397位点磷酸化是必要条件,而且RhoA/ROCK、细胞骨架和FAK相互影响并协同调节械拉伸诱导hMSCs向肌腱细胞方向分化。本研究对认识机械拉伸诱导hMSCs定向分化的分子基础以及分化过程中力信号传导具有重要意义,还可为临床上腱/韧带创伤的治疗及其组织工程化提供定量的参考依据。
Tendon/ligament is a kind of connective tissue subjected to complicated mechanical microenvironment which is controlling their growth, development and regeneration. However, it is always injured in the daily life, and the cure of the injuries is a serious challenge for clinician.
Mesenchymal stem cells (MSCs) are able to differentiate into a lot of cell lineages. For their advantages, such as abundant source, fast proliferation, easy to transfer exogenous gene, low immune reaction, they become one of the research focus of tissue engineering, gene therapy or cell therapy. At present, it was reported that MSCs were able to differentiate into tenocytes by mechanical stimuli, and to be used for tendon/ligament repair. It brings new hopes for the appliacation of tissue engineering for tendon/ligament repair. However, the mechanical stretch-induced tenogenic differentiation is not well understood and the mechanotransduction mechanism is still unclear. Therefore, it becomes an important research content of in the furthure.
This study further investigated the effect of mechanical stretch on tenogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), and the mechanotransduction mechanism during the process. To search for an appropriate stretch parameter, cyclic stretch device (ST-140) was used to load mechanical stretch on hMSCs which growth on febronectin-coated silicon chambers. After stretch, tendon-related gene and protein expressions were analyzed by real-time RT-PCR and western blot. After that, to examine the effect of RhoA/ROCK, cytoskeletal organization and FAK (Focal adhesion kinase) during mechanical stretch-induced tenogenic differentiation, Y-27632, ctochalasin D and PF 530228 (PF 228) were used to inhibit RhoA/ROCK, cytoskeletal organization and FAK, respectively.
The main experiments and major results are as follows:
①hMSCs multi-lineages differentaiton potential
To examine the multi-lineages differentaiton potential of hMSCs used in this study, hMSCs were cultured in OS (osteogenic) induction media, AD (adipogenic) induction media or CH (chondrogenic) induction media for 21 d. Alizarin red S, oil red O and acian blue staining were conducted to analyzed the OS induction, AD induction and CH induction, respectively. The staining results suggest that hMSCs could differentiate into osteogenic, adipogenic and chondrogenic.
②Mehcanical stretch-induced tenogenic differentiation of hMSCs
Aimed to search for appropriate stretch parameters, hMSCs were loaded with different mechanical stretch (0.1 Hz, 15% strain, 24 h and 48 h; 0.1 Hz, 10% strain, 24 h and 48 h; 0.1 Hz, 5% strain, 24 h and 48 h; 1.0 Hz, 15% strain, 24 h and 48 h; 1.0 Hz, 10% strain, 24 h and 48 h; 1.0 Hz, 5% strain, 24 h and 48 h), gene expressions of collagen type I (Col I), collagen type III (Col III), tenascin-C (TNC) and scleraxis (SCX), and protein expressions of Col I and TNC, were anaylized by real-time RT-PCR and western blot. The results showed that only the mechanical stretch (1.0 Hz, 10% strain, 24 h and 48 h) can promote the gene expressions of Col I, Col III, TNC and SCX,and the protein expressions of Col I and TNC. It suggests that 1.0 Hz, 10% strain mechanical stretch can induce tenogenic differentiation of hMSCs. Concomitant with this process, hMSCs were induced to realign vertically to the direction of stretch, and the trend of realignment was dependent on the strain, frequence and duration of mechanical stretch.
③Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in morphology of hMSCs
Y-27632, cytochalsin D and PF 228 had important effect on mechanical stretch-induced changes in cytoskeletal dynamics which defines cell shape. The changes in density of actin fibers and vertical alignment of actin fibers to the direction of stretch, are due to the adaptation to mechanical loads which can resist and reduce experienced mechanical force. Inhibition of RhoA/ROCK resulted in lacking actin fibers, failure in trailing-edge retraction and leaving a long tail behind cells, and showed stellate-like morphology. However, in the presence of Y-27632 and mechanical stretch resulted in discontinuous actin fibers and very short actin fibers in the cell edges, and no significant changes in cell shape and favorite alignment with respect to the direction of stretch. Cytochalasin D treatment resulted in disruption of actin fibers organization and circular cell shape. When the cytochalasin D treated cells were exposure to stretch, the actin fibers were further disrupted and the cell shape became more circuler. PF 228 incubation resulted in more circular cell shape and shorter actin fibers. Interestingly, PF 228 incubation with mechanical stretch resulted in stellate-like cells, shorter actin fibers and rough edges, and even resulted in generation of cell debris. In addition, there were also no specific alignments of cells and actin fibers with respect to the direction of stretch. These data suggest that mechanical stretch facilitates actin fibers formation and induces realignment of cells by inducing actin fibers perpendicular aligning to the direction of stretch which can resist and adapt to the force balance. And the stretch-induced realignment is needed activation of RhoA/ROCK, intact cytoskeltal organization and FAK phosphorylation.
④Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in FAK phosphorylation and tenogenic differentiation of hMSCs
Western blot showed that with experience of stretch for 30 min, phosphorylation of FAK at Tyr397 was increased up to 1.966±0.167 times. When cells were treated by Y-27632, phosphorylation of FAK at Tyr397 was decreased to 1.081±0.1 times. When the assembling of F-actin was blocked by cytochalasin, phosphorylation of FAK at Tyr397 was decreased to 1.095±0.099 times. Treatment with PF 228 resulted in dramatical decrease of phosphorylation of FAK at Tyr397. It was reduced to 0.395±0.075 times. It indicates RhoA/ROCK and cytoskeletal organization are essential to mechanical stretch-activated FAK phosphorylation.
Except for the influence on morphology and stretch-activated FAK phosphorylation, Y-27632, cytochalsin D and PF 228 have important effect on gene expressions and even to differentiation. Y-27632, cytochalsin D or PF 228 inhibited the mechanical stretch-induced gene expressions of Col I, Col III, TNC, SCX, EphA4, Eya2 and Six1, and the protein expressions of Col I, Col III and TNC. It suggests that RhoA/ROCK, cytoskeleton and FAK are necessary for mechanical stretch-induced tenogenic diferentiation of hMSCs. Moreover, RhoA/ROCK, cytoskeletal organization and FAK interacted with each other in the stretch-induced tenogenic differentiation of hMSCs.
In summary, we can infer that RhoA/ROCK, cytoskeletal dynamics and FAK interacte with each other and compose a signaling network to sense mechanical environment. The signaling network converts mechanical stretch into biochemical signaling, and then triggers stretch-induced tenogenic differentiation. Inhibiting any component of the signaling network, it fails to convert mechanical stretching into biochemical signaling and the stretch-induced tenogenic differentiation will be aborted. It provides novel view into the mechanisms of stretch-induced tenogenesis and supports the therapeutic potential of hMSCs.
引文
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