CaN-NFAT信号通路在应力介导的成肌细胞凋亡中的作用及其作用机制

发布时间：2018-03-15 08:22

  本文选题：周期性张应力　切入点：成肌细胞　出处：《青岛大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：颌面部肌肉组织的适应性改建在正畸功能矫形中都发挥了重要作用。成肌细胞是构成颌面部肌肉组织的重要组成部分,是适应性改建的主要体现者。目前多数学者的研究集中于成肌细胞的分化、增殖等方面,忽视了成肌细胞凋亡方面的研究。而成肌细胞的凋亡与成肌细胞的分化、增殖一样,在颌面部肌肉组织的生长和改建中发挥着重要的作用。本研究通过构建成肌细胞力学刺激模型研究周期性张应力对大鼠L6成肌细胞凋亡的影响,明确CaN-NFAT信号通路在应力介导的大鼠L6成肌细胞凋亡中的作用,从而阐明功能矫形时成肌细胞适应性改建的分子机制,为正畸医生治疗错颌畸形提供理论支持。 方法：本实验应用多通道细胞牵张应力加载系统对大鼠L6成肌细胞构建的力学刺激模型分别施加Oh、1h、2h、6h、12h、24h的周期性张应力(0h组即为对照组),加载的力值为15%的细胞形变率；加载的频率为10个循环/min,每个循环均包括3s拉伸和3s松弛；于倒置相差显微镜下对成肌细胞的形态学改变和生长状况进行观察；经过Hoechst33258荧光染料染色后,采用荧光显微镜观察凋亡的成肌细胞；采用流式细胞术分别分析各组成肌细胞的凋亡率；运用实时荧光定量PCI技术及Western blot技术分别检测周期性张应力作用下各组成肌细胞钙调神经磷酸酶(CaN)及NFAT的mRNA和蛋白含量；采用CaN的特异性抑制剂环孢素(CsA)明确CaN在周期性张应力介导的L6成肌细胞凋亡中的作用,从而进一步明确内质网应激与应力介导的大鼠L6成肌细胞凋亡之间的关系。采用统计学软件SPSS17.0对所得的数据进行统计学分析。 结果： 1大鼠L6成肌细胞在受到大小为15%细胞形变率,频率为10个循环/min的周期性张应力后,贴壁生长情况良好,细胞无变性并且脱落率极低,说明成肌细胞体外培养-力学刺激模型构建成功； 2当大鼠L6成肌细胞受到长时间的周期性张应力作用后,细胞排列方向由原先的杂乱无章变为顺应力场方向排列,并且随着应力作用时问变长,细胞排列方向变得更加明显； 3Hoechst33258荧光染色及流式细胞术的检测结果显示15%的细胞形变率,10个循环/min的周期性张应力诱导大鼠L6成肌细胞发生凋亡,并且在一定的作用时间范围内,成肌细胞的凋亡率随着应力作用时间的延长而升高,在24h达到高峰； 4实时荧光定量PCR的检测结果显示,随着应力作用时间的延长,钙调神经磷酸酶(CaN)及NFAT的mRNA表达量均有所增加；Western blot的检测结果显示,随着应力作用时间的延长,NFAT的蛋白量也有所增加,这与PCR的检测结果相符；可见,CaN-NFAT信号通路参与了周期性张应力作用下的大鼠L6成肌细胞的力学信号传导。 5CaN的特异性抑制剂环孢素(CsA)抑制CaN活性后,成肌细胞的凋亡率下降,钙调神经磷酸酶(CaN)和NFAT的mRNA表达量、NFAT的蛋白量下降,差异具有统计学意义(P0.05)。 结论： 1.15%的细胞形变率,10个循环/min的周期性张应力可以诱导大鼠L6成肌细胞发生凋亡； 2. CaN-NFAT信号通路可能参与周期性张应力介导的大鼠L6成肌细胞凋亡。
[Abstract]:Objective: facial muscle tissue remodeling in orthodontic orthopedic have played an important role. Myoblasts constitute an important part of facial muscle tissue, is the main embodiment of adaptive remodeling. At present many scholars study focused on the differentiation of myoblast proliferation, etc., ignored the study muscle cell apoptosis and differentiation. The apoptosis of muscle cells, and the proliferation of skeletal muscle cells, plays an important role in the growth and reconstruction of maxillofacial muscle tissue. This study builds the myoblast mechanical stimulation effects of myoblasts on apoptosis of rat L6 model of cyclical stretch clearly, the CaN-NFAT signaling pathway of myoblast apoptosis in stress mediated L6 in rats, so as to elucidate the molecular mechanism of functional orthopedics into rebuilding muscle cell adaptability, for treatment of malocclusion orthodontic doctors Provide theoretical support.
Methods: the application of multi channel cell traction mechanical tensile stress loading system of myoblast construction on rat L6 stimulation model was applied to Oh, 1H, 2h, 6h, 12h, periodic Zhang Yingli 24h (0h group as the control group), the loading force value for cell deformation rate of 15%; loading the frequency of 10 cycles of /min, each cycle includes 3S stretch and 3S relaxation; in inverted on the morphology of myoblast changes and growth status were observed under microscope; after Hoechst33258 fluorescent dye staining, observation of myoblasts apoptosis by fluorescence microscope; the apoptosis rate of the muscle cells were analyzed by flow cytometry FCM; using real-time fluorescence quantitative PCI and Western blot technology were used to detect the cyclic tensile stress under the action of the muscle cells of calcineurin (CaN) and mRNA protein content and NFAT; the specificity of CaN suppression Preparation of cyclosporine A (CsA) clear CaN myoblasts apoptosis in periodic Zhang Yingli mediated by L6, so as to further clarify the endoplasmic reticulum stress and stress mediated the relationship between rat L6 muscle cells apoptosis. Statistical analysis was performed using SPSS17.0 statistical software for the data.
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本文编号：1615211