P120-catenin在张力诱导终板软骨细胞炎症中的调控作用

发布时间：2018-03-29 14:12

本文选题：终板软骨细胞　切入点：间歇性循环牵张力　出处：《皖南医学院》2017年硕士论文

【摘要】：目的:体外培养建立大鼠终板软骨细胞张力诱导炎症模型,探讨NF-κB信号通路与P120-catenin在张力诱导终板软骨细胞炎症中的调控机制。方法:无菌条件下取出大鼠的腰椎终板软骨,胶原酶消化法提取终板软骨细胞,细胞培养至二代后,采用FX-5000细胞应变加载系统体外诱导构建终板软骨细胞张力载荷下的炎症模型;采用特异性抑制剂Bay117082调控NF-κB信号通路;通过基因转染过表达P120-catenin基因。实时RT-PCR检测加力前后软骨炎症因子MMP3,MMP9,MMP13,COX-2,iNOS等基因的表达变化;AlamarBlue法检测终板软骨细胞增殖率、流式细胞术检测细胞凋亡,应用鬼笔环肽染色检测细胞加力前后骨架的变化;免疫荧光法检测P120-catenin在终板软骨细胞胞中的表达与定位;核质分离及蛋白免疫印迹检测胞核与胞质中NF-κB信号通路相关蛋白P65、p-P65、IkBα、p-IkBα表达变化;Dual-Luciferase报告基因检测NF-κB信号通路激活状态。结果:张力加载会诱导终板软骨细胞发生炎症性反应,相关炎症基因的表达上调。张力刺激不影响终板软骨细胞的增殖,不会诱导细胞的发生凋亡,但会使软骨细胞骨架发生一定程度的形态改变。随着张力加载的进行,终板软骨细胞中NF-κB信号通路激活,加力时间延长后炎症相关因子的表达明显增高;抑制NF-κB信号通路后,相关软骨细胞炎症因子表达降低,终板软骨细胞炎性症状减轻。张力载荷下终板软骨细胞中P120-catenin在基因和蛋白水平的表达均明显降低,质粒转染过表达P120-catenin后,软骨细胞中P120-catenin在基因和蛋白水平表达显著增高,张力刺激NF-κB信号通路激活被抑制,张力诱导的终板软骨细胞炎性症状明显减轻。结论:张力载荷通过诱导NF-κB信号通路的激活而导致终板软骨细胞炎症反应;这种调控是由于张力刺激下调软骨细胞质内的P120-catenin,减弱了P120-catenin对NF-κB信号通路的抑制作用,最终导致终板软骨细胞的炎症反应。调控NF-κB信号通路及P120-catenin的表达能够在很大程度上减轻终板软骨的炎性症状。
[Abstract]:Objective: to establish the in vitro tension of rat endplate chondrocyte induced inflammation model of NF- B signaling pathway and P120-catenin in tension induced regulation mechanism of endplate chondrocyte inflammation in rats. Methods: the lumbar endplate cartilage removed under sterile conditions, extraction of endplate cartilage cells by collagenase digestion, the cells were cultured for two generations, the by constructing inflammation model of endplate chondrocytes tension loading FX-5000 cell strain loading system in vitro; the specific inhibitor of Bay117082 regulation of NF- B signaling pathway by gene transfection; expression of P120-catenin gene. After real-time RT-PCR for the detection of inflammatory factors MMP3 cartilage afterburner MMP9, MMP13, COX-2, and the expression change of iNOS gene; detection of endplate cartilage cell proliferation AlamarBlue assay, flow cytometry, cell skeleton change detected before and after the application of phalloidin staining afterburner; The expression and localization of immunofluorescence was used to detect P120-catenin in the cytoplasm of endplate chondrocytes; NF- kappa B signaling pathway related protein P65, cell separation and detection of nuclear protein nuclear and cytoplasmic p-P65, IkB alpha, alpha p-IkB expression; Dual-Luciferase reporter gene detection of NF- kappa B signaling pathway activation. Results: tension the loading will induce inflammatory reaction of endplate cartilage cells, upregulation of inflammatory gene expression. The tension did not affect the endplate cartilage cell proliferation, apoptosis does not induce cells, but the change will make a certain degree of cartilage cytoskeleton morphology. With tension loading, NF- kappa B signaling pathway of endplate chondrocytes. After long time, boosting of inflammatory cytokines expression was significantly increased; the inhibition of NF- B signaling pathway and related inflammatory factors decreased expression of cartilage cells, cartilage endplate cells inflammatory symptoms Reduce the expression of P120-catenin. Endplate chondrocytes tension loads in gene and protein levels were significantly decreased after overexpression of P120-catenin plasmid transfection, expression of P120-catenin in cartilage cells in gene and protein levels increased significantly, tension stimulation of NF- B signaling pathway activation is inhibited, the tension induced by endplate chondrocyte inflammatory symptoms significantly reduced. Conclusion: the result of endplate cartilage cell inflammatory response induced by activation of NF- tension load B pathway; this regulation is stimulated by cartilage due to tension in the cytoplasm of P120-catenin, weaken the inhibitory effect of P120-catenin on NF- B signaling pathway, resulting in inflammation of endplate cartilage cells. The expression of B signaling pathway and regulation of NF- P120-catenin can reduce the endplate cartilage largely inflammatory symptoms.

【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.53

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