【作者】 谭家莉；

【导师】 段银钟； Prof.OphirKlein； 袁晓；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2010， 博士

【摘要】 口腔颌面部的畸形严重影响患者的身心健康和家庭幸福,功能矫治成为重要的治疗手段。面颌部肌肉组织的适应性变化在牙颌面畸形的矫治中扮演着重要角色。探讨矫形力对肌肉细胞的增殖、分化、凋亡的具体影响和分子机制,对进一步认识矫形治疗的分子机制以及合理利用矫形力提高临床预后具有重要意义。既往研究表明,应力条件下ROS生成显著增加,而JNK、NFkappaB也具有不同程度的活化,提示它们之间可能存在一定的联系。而它们之间如何交互对话以及如何影响细胞的命运尚不清楚。本研究旨在阐明机械牵张力条件下,ROS是调节JNK和NFkappaB这两条信号通路间的交互作用的分子机制及其对细胞功能的具体影响。【目的】本研究旨在探索不同应力条件下ROS的动态变化,他们如何影响成肌细胞的增殖、分化和凋亡,NFkappaB和JNK在该过程中的作用;从而获得应力调控细胞凋亡的相关信息,为正畸治疗中合理使用应力引起肌肉改建提供相应的理论依据。【方法与结果】(1)不同幅度的周期性应力对成肌细胞增殖、分化、凋亡的影响不同。本课题首先通过MTT和DNA Ladder实验测定在受到不同大小的牵张应力作用下(0%, 5%, 10%, 15%, 20% /10 cycles / min)的C2C12细胞的增殖和凋亡的改变,通过检测细胞分化相关基因的表达改变,分析应力条件下细胞分化状态的改变。结果发现,5%的周期性应力可以促进细胞的增殖,同时抑制分化培养基诱导的细胞分化相关基因myogenin等的表达。当牵张应力大于10%后,可以观察到明显的DNA断裂。除此以外,我们还发现牵张应力大于10%后,细胞内开始出现大量的PARP剪切产物,随应力的增加而增多。当周期性牵张应力超过15% 10cycles/min时,整体存活细胞数明显降低。由此可见,高强度牵拉介导的细胞凋亡,参与整体细胞数的减少,可能不利于组织的重塑和再生。(2)周期性应力对ROS的产生、NFkappaB活性、JNK1活化的影响。我们通过荧光染料检测ROS的生成,荧光报告系统检测NFkappaB的活性以及Western Blot检测磷酸化和总体JNK1的水平;分析不同应力条件下细胞内ROS产生量、JNK和NFkappaB的活化程度以及它们间的相关性。结果发现,随着周期性应力的增加,细胞内ROS生成逐渐增加。随着应力的增大,细胞的凋亡逐渐增加,与此同时,JNK1活性逐渐增高,而JNK1表达水平并没有明显改变。与ROS生成量和JNK1活化逐渐增加不同,应力在10%以下时NFkappaB的活性随着应力的增大而增大,而当应力大于15%时,NFkappaB活性开始下降。为了阐明ROS和JNK活性二者之间的关系,我们提前4小时对细胞使用不同浓度的ROS清除剂NAC,然后对细胞施加20%/10cycles/min的牵张应力24小时。结果发现,ROS的产生被NAC抑制。当NAC的浓度达到100μM时,ROS降至基线水平。10μM NAC开始抑制JNK的活性,随着NAC的浓度的增加,JNK活性逐渐降低。10μM NAC处理后增加NFkappaB的活性。NAC浓度的继续增加,NFkappaB的活性先升高后降低,当NAC升高至1000μM时,NFkappaB的活性降至基线以下水平。所有这些结果提示,当ROS的量积累到一定的时候,JNK开始激活,而ROS的量较低的时候,NFkappaB被激活。(3)低强度应力条件下NFkappaB的活化,促进miR146a的表达,后者抑制细胞分化。鉴于低强度条件下,NFkappaB活化,细胞增殖增加而分化受阻。我们分析了该条件下,NFkappaB的重要下游靶分子miR146a的表达及其在过程中的作用。采用miRNA特异性qRT-PCR我们发现,低强度应力条件下,miR146a表达增加,过表达miR146a可以抑制细胞的分化,抑制miR146a的功能可以促进细胞的分化。借助生物信息学发现,分化重要的调节基因Numb是miR146a的靶分子,而过表达miR146a可以观察到Numb的表达降低。Numb的3’UTR报告系统,显示miR146a可以抑制Numb的3’UTR的活性。(4)高强度应力条件下,ROS大量产生,JNK1的激活抑制NFkappaB的活化。在较强的应力作用下,NFkappaB和JNK的活性变化呈现相反的关系,提示JNK的活性可能引起了NFkappaB活性的降低。因此,我们使用JNK的抑制剂预先处理细胞或者JNK的特异性RNAi 24小时后施加20%的周期性的牵张力24小时。结果发现,与对照组相比,抑制JNK信号会增加NFkappaB的活性,进而增强细胞的存活。为了明确NFkappaB活性的抑制是否对JNK激活引起的细胞凋亡起关键作用,我们在JNK被抑制的情况下进一步利用NFkappaB的阻断剂aspirin。结果发现,与单纯使用JNK抑制剂组相比,联合使用JNK和NFkappaB的抑制剂显著降低高强度应力条件下细胞活力,说明在高强度应力条件下,JNK活化通过抑制NFkappaB引起细胞凋亡。(5)高强度应力条件下,JNK的激活阻断了NFkappaB的核转位和Bcl2的转录。为研究JNK1抑制NFkappaB的活化的详细机制和下游效应,我们进一步通过分离胞浆胞核,分析NFkappaB的核定位及RT-PCR分析其下游促进细胞存活分子的表达。结果发现,20%牵张应力条件下,细胞核内几乎检测不到的p65定位。不仅如此,20%的牵张应力条件下NFkappaB的下游抗凋亡靶基因Bcl2几乎不表达;阻断JNK的活性引起细胞核中的NFkappaB水平升高,Bcl2的表达也得以恢复。不仅如此,我们还观察到,JNK抑制剂的功能强于JNK1 siRNA,提示该过程中JNK的其他亚型也可能参与其中。【结论】低强度的应力,促进细胞的增殖、抑制细胞的分化;而高强度应力则引起成肌细胞的凋亡。该条件下,ROS的生成引起NFkappaB和JNK活性的动态变化。低强度条件下,NFkappaB活化引起miR146a的表达增加,后者抑制Numb的表达,进而抑制细胞的分化;而高强度应力条件下,JNK活化,抑制NFkappB的活性,引起抗凋亡基因的表达下调,细胞凋亡增加。该研究为合理应用应力,进行矫形治疗提供了重要参考。更多还原

【Abstract】 Mechanical force is essential for orthodontics, in which the adjustment of the soft tissue, especially the muscle is the key to the treatment. During the treatment, Mechanical forces induce changes in the structure, composition, and function of tissues such as bone, vessel and muscle. Molecularly, the mechanical force signal is transduced into the cell, resulting in the cell proliferation, differentiation and apoptosis, which is vital for the adjustment.Recently, responses to stretch have been intensely studied in load-sensitive cells, which include fibroblasts, osteoblasts, endothelial cells, smooth muscle cells and skeletal muscle cells. Apoptosis has been found to be induced in myocytes by inappropriate mechanical stimuli under pathological conditions or during clinical treatments. Although, apoptosis is intensively studied in muscle tissue and muscle cells, most of those studies just focus on muscle cell apoptosis in atrophy. The mechanisms responsible for muscle cell apoptosis under stretches are rarely studied.ROS is abundantly induced in muscle cells by stretch which is one of the key factors responsible for apoptosis and related muscle damages. Up to now, how ROS regulate JNK and NFkappaB discriminately and coordinately is poorly understood. Since there should be a coordinated regulation of these two pathways, it is of great value to elucidate how ROS regulate the crosstalk between NFkappaB and JNK pathway under the mechanical forces.[Aims] The present study explores the effects of different mechanical forces on cellfunction, and the dynamic changes of ROS under different mechanical forces and how they are involved in determination of myoblast fate and integration of both NFkappaB and JNK signal pathway.[Methods and results](1) Correlation between cyclic stretch magnitudes and cell fate.To study the detailed effects of the cyclic stretches of a serial of magnitudes on cell growth and differentiation, we first evaluated cell viability upon serial cyclic stretches (0%, 5%, 10%, 15%, 20% /10 cycles / min). When the extension magnitude is about 5%, cell growth was facilitated. In contrast, myogenic medium induced cell differentiation under this condition was inhibited as seen by myogenin expression. With the extension magnitudes exceeding 15% 10cycles/min, cell viability decreased. Since both proliferation and cell death affected the MTT results, we then tested whether apoptosis was involved in the process. To this end, DNA fragmentation analysis was applied. Consistent with the decreased cell viability, we observed obvious DNA fragmentation under cyclic stretches of over 10% extension, suggesting apoptosis contributed at least partially to the observed cell viability. In addition, we analyzed the cleavage of PARP, another marker of apoptosis, which has been shown in muscle cell apoptosis. Consistently, abundant PARP cleavage was observed when the cyclic stretches were over 15%.(2) Relationship among ROS production, NFkappaB activity and JNK1 activation.Considering the paradoxical roles of JNK and NFkappaB in ROS induced apoptosis, we examined ROS generation, NFkappaB activity and phosphorylated JNK1 levels under serial stretches. With the stretch magnitudes increased, consistent with the increased apoptosis, JNK1 were gradually activated, with no obvious changes of JNK1 expression. In contrast, NFkappaB activity increased with the extension rate increasing within the magnitudes no more than 10%, and it began to decline when the extension rate is over 15%. To confirm the relationship between ROS and JNK activation, we pre-treated cells with ROS scavenger NAC at serial concentrations 4 hrs before cells undergoing a 24 hrs of 20%/10cycles/min stretch. ROS production was significantly inhibited by NAC treatment, with the ROS level decreased to nearly the baseline when the NAC concentration was 100μM. 10μM NAC treatment began to inhibit the JNK activation, and with the NAC concentration increased, JNK activation decreased gradually. In contrast, 10μM NAC treatment increased the activity of NFkappaB. With the NAC concentration higher, NFkappaB activity began to decline from the climax, and became lower than the baseline when NAC concentration was 1000μM. All of these suggested that JNK activation occurred only when ROS production accumulated sufficiently while NFkappaB was activated at a much lower threshold of ROS abundance.(3) Low magnitude of cyclic stretch increased the expression of miR-146a in differentiating C2C12 cellsFrom the above data, we deduce that low magnitude of cyclic stretch may affect cell differentiation. To this end, we examined cyclic stretch effects on differentiation. Remarkably, cyclic stretch reduced the expression of myogenin, an indicator of differentiation. Then, we examined the expression level of miR-146a, which is a known target of NFkappaB, by miRNA qRT-PCR. Strikingly, compared with the control group, miR-146a in cyclic stretch treated group was nearly 4 folds higher. miR-146a inhibited Numb expression through its 3’UTR. To elucidate the potential role of miR-146a in muscle differentiation and proliferation, putative targets of this miRNA were predicted by the online tool Targetscan 4.2. There are nearly 200 potential targets of miR-146a, among which Numb has been reported to be involved in myogenesis. To confirm the regulation of Numb by miR-146a, we then analyzed the expression of Numb in differentiating C2C12 cells under cyclic stretch. Consistent with the increase of miR-146a, we found a decline of Numb protein level after cyclic stretch. Furthermore, transfection of siRNA-like miR-146a also repressed the expression of Numb in C2C12 cells.(4) Under high mechanical forces, JNK1 activation contributed to the NFkappaB inhibition upon excessive ROS generation.Usually, ROS are the inducer of NFkappaB activation. Strikingly, in contrast to the continual increase of activated JNK1, here we did not observe a steady increase of NFkappaB activity with ROS gradual accumulation. NFkappaB activity began to decline from the climax when the magnitudes of stretch exceeding 15%. The reverse relationship between NFkappaB and JNK at intensive stretches prompted us to assume that JNK activation might contribute to the decline of NFkappaB activity. To this end, we pretreated cells with a specific JNK chemical inhibitor or specific RNAi 24 hrs before cells undergoing a 24hrs of 20% cyclic stretch. Using the RNAi technique, we efficiently reduced the expression of JNK1 to about 30%. Compared with the control, inhibition of JNK signal increased the NFkappaB activity, and consistently cell viability increased. To confirm whether inhibition of NFkappaB activity was critical for the cell death induced by JNK activation, we further blocked the activation of NFkappaB by its inhibitor aspirin in combination with JNK inhibition. 5mM aspirin nearly blocked the activation of NFkappaB as indicated by the luciferase assay. Upon 20% stretch, compared with the group treated with JNK inhibitor only, combination of JNK inhibitor and NFkappaB inhibitor decreased the cell viability significantly, suggesting that NFkappaB inhibition played an important role in JNK activation induced cell death.(5) Under high mechanical forces, JNK1 activation blocked the nuclear translocation of NFkappaB and the transcription of Bcl2.Since nuclear translocation and DNA binding ability are the two main factors affecting NFkappaB transcriptional activity, we firstly analyzed the nuclear levels of p65 under different conditions. Nuclear p65 was rarely detectable upon 20% extension stretch. In contrast, blockade of JNK activity by RNAi or chemical inhibitor rescued the nuclear localized p65. Furthermore, we observed the expression of Bcl2, which is one of the most important anti-apoptotic target genes of NFkappaB. Upon 20% extension stretch, consistent with the increase of nuclear NFkappaB by JNK blockade, Bcl2 expression was upregulated. Furthermore, we could see that the chemical inhibitor of JNK functioned stronger than JNK1 siRNA, which might be explained by the fact that other JNK isoforms were inhibited simultaneously by the chemical inhibitor or alternatively the knockdown efficiency in our study was compromised. Using the cyclic stretch model, we observed that ROS generation accumulated progressively and cell death occurred under excessive stretches. NFkappaB transcriptional activity increased when there was minor to moderate ROS production, and began to decline when ROS generation continued. In contrast, obvious JNK activation just occurred when ROS generation was abundant enough under severe stretches. Through blocking JNK activation by a specific chemical inhibitor or JNK1 specific RNAi, we concluded that JNK activation was responsible for the inhibition of NFkappaB activity, which subsequently functioned importantly in apoptosis.【Conclusion】In this study, we serially studied the effects of cyclic stretch on cell proliferation, differentiation and apoptosis. We found that under low magnitude mechanical forces, cell growth was facilitated while cell differentiation was inhibited. In this process, NFkappaB activation and miR146a expression functions importantly. Under high magnitude mechanical forces, increased ROS induced by mechanical forces activates JNK1, which in turn repress NFkappaB and its target Bcl2, resulting in cell apoptosis. Our observation here may facilitate our manipulation of mechanical forces in Sports Medicine and Ortho Medicine.更多还原