RANKL-NFκB-ADAMTS5信号轴调控软骨退变的机制研究

发布时间：2018-03-04 04:22

本文选题：RANKL　切入点：NFκB　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:明确核因子K B受体活化因子配体(Receptor Activator for Nuclear Factor-κ B Ligand,RANKL)对软骨的直接作用及作用机制,为关节软骨退变的治疗提供新思路。方法:体外细胞实验选用软骨细胞株ATDC5,以明确RANKL对软骨细胞的直接作用。体外培养采用牛软骨片,以明确RANKL对软骨组织的直接作用。体内动物实验采用C57BL/6J小鼠,以明确RANKL在体内对关节软骨的作用。第一部分:RT-PCR检测正常软骨细胞中核因子κ B受体活化因子(Receptor Activator for Nuclear Factor-κ B,RANK)和 RANKL 的表达,RT-qPCR 检测软骨细胞ATDC5在分化过程中Col2a1、Co110a、OPG、SOX9、RANK和RANKL的时空表达,Western蛋白印迹检测RANKL刺激后ATDC5细胞中RANK的表达变化,HE、Alcian Blue和Safranin 0染色及Markin评分分析RANKL刺激对软骨细胞或组织的直接作用。第二部分:RT-qPCR 检测 RANKL 刺激后 ATDC5 细胞 Col2a1、MMP9、MMP13、ADAMTS5的时空表达变化,Western蛋白印迹检测RANKL刺激后MMP13、ADAMTS5的时空表达变化,不同浓度的RANKL刺激对ADAMTS5表达的影响,及RANKL刺激0～120分钟内总IKB-α和磷酸化的IKB-α的表达变化。免疫荧光和激光共聚焦观察p65的入核情况。IKB-α特异性抑制剂选用BAY11-7082。RANK沉默选用小干扰RNA。第三部分:Micro-CT检测关节腔RANKL后胫骨骨体积分数的变化,RT-qPCR检测股骨TRAP、Col2a1和Col10a的表达情况,HE染色分析关节软骨的形态变化情况,免疫组织化学染色检测Col2a1、Col10a、MMP9、MMP13、OPG、RANKL和ADAMTS5等蛋白的表达变化。采用SPSS17.0软件包对数据进行统计学分析。结果:1)软骨细胞可以表达RANK及RANKL。在软骨分化过程中,RANKL和RANK的表达随时间增多。外源性RANKL刺激可以使软骨细胞的RANK表达上调。外源性的RANKL刺激可导致软骨结构紊乱,蛋白多糖丢失,Markin评分升高(P0.05)2)相对于对照组,RANKL刺激ATDC5细胞后,与软骨退变相关的MMP9、MMP13和ADAMTS5的表达显著升高,且有浓度依赖性;而软骨标志因子II型胶原和X型胶原的mRNA水平显著下降(P0.05)。RANKL刺激后细胞中总IKB-α的表达随时间表达下降,磷酸化IKB-α开始随时间表达增多,在30min表达达到峰值,随后下降。免疫荧光染色后,激光共聚焦观察到P65在30min时有明显的入核。加入IKB-α特异性抑制剂BAY11-7082后,再次加入RANKL刺激ADAMTS5的表达增量随着抑制剂浓度增加而下降。RANK沉默后,ADAMTS5的上调也不再观察到。3)关节腔内注射RANKL可以引起骨体积分数下降、Col2a1和Col10a的表达下降及TRAP的表达升高。HE染色显示软骨层空泡的出现及软骨细胞数目的减少。免疫组织化学染色显示,与软骨标志蛋白Co12a、Col10a的表达下降,而与软骨退化相关的ADAMTS5、MMP9、MMP13表达升高。与此同时,RANKL表达升高,而OPG的表达下降。结论:RANKL可以通过NFκ B信号通路来上调ADAMTS5的表达,直接导致软骨退行性变。因此,可以以RANKL-NFκB-ADAMTS5信号轴为干预靶点,作为预防和治疗关节软骨退变的新途径。
[Abstract]:Objective: to investigate the direct effect and mechanism of nuclear factor K B receptor activating factor ligand Receptor Activator for Nuclear Factor- 魏 B Ligand RANKL on cartilage. Methods: the chondrocyte line ATDC5 was selected in vitro to determine the direct effect of RANKL on chondrocytes. Bovine chondrocytes were cultured in vitro. In order to clarify the direct effect of RANKL on cartilage tissue, C57BL / 6J mice were used in animal experiment in vivo. In order to clarify the effect of RANKL on articular cartilage in vivo. The first part was to detect the expression of nuclear factor 魏 B receptor activating factor (Receptor Activator for Nuclear Factor- 魏 B RANK- 魏 B) and RANKL in normal chondrocytes by RT-qPCR. The changes of RANK expression in ATDC5 cells after RANKL stimulation were detected by Western blot and Markin scores were used to analyze the direct effect of RANKL stimulation on chondrocytes or tissues. Part 2: RT-qPCR was used to detect ATDC5 after RANKL stimulation. The temporal and spatial expression of ADAMTS5 in Col2a1 / MMP9 / MMP13 / MMP13 was detected by Western blot, and the expression of ADAMTS5 was detected by Western blot after RANKL stimulation, and the expression of ADAMTS5 was detected by Western blot. Effects of different concentrations of RANKL stimulation on ADAMTS5 expression, The expression of total IKB- 伪 and phosphorylated IKB- 伪 was observed by immunofluorescence and laser confocal focusing. The specific inhibitor of IkB- 伪 was BAY11-7082.RANK silencing and small interference RNA was selected by BAY11-7082.RANK silencing. Part 3: Micro-CT was used to detect RANKL in articular cavity. The changes of tibial bone volume fraction and the expression of TRAPL Col2a1 and Col10a in femur were detected by RT-qPCR. The morphological changes of articular cartilage were analyzed by HE staining. Immunohistochemical staining was used to detect the expression of RANKL and ADAMTS5 proteins in Col2a1 / Col10aOMMP9 / MMP13OPGnL. The data were analyzed statistically by SPSS17.0 software package. Results: 1) chondrocytes could express RANK and RANKL. the expression of RANKL and RANK in chondrocytes during chondrogenic differentiation was observed over time. Exogenous RANKL stimulation can up-regulate RANK expression in chondrocytes. Exogenous RANKL stimulation can lead to cartilage structure disorder. Compared with the control group, the expression of MMP9, MMP9, MMP13 and ADAMTS5, which were associated with cartilage degeneration, was significantly increased in a dose-dependent manner. However, the mRNA level of type II collagen and type X collagen decreased significantly after stimulation with P0.05. RANKL. The expression of total IKB- 伪 decreased with time, and the expression of phosphorylated IKB- 伪 began to increase with time, and reached its peak at 30 minutes. After immunofluorescence staining, laser confocal observation showed that p65 had obvious nucleation at 30min. After adding IKB- 伪 specific inhibitor BAY11-7082, The expression increment of ADAMTS5 stimulated by RANKL again decreased with the increase of inhibitor concentration. After rank silencing, the upregulation of ADAMTS5 was also no longer observed. 3) Intraarticular injection of RANKL could induce the decrease of bone volume fraction and the expression of Col2a1 and Col10a and the surface of TRAP. He staining showed the appearance of chondrocyte vacuoles and the decrease of chondrocytes. Immunohistochemical staining showed that, The expression of ADAMTS5, MMP9, MMP13, which is associated with cartilage degeneration, decreased, while the expression of RANKL increased, while the expression of OPG decreased. Conclusion the expression of ADAMTS5 can be upregulated by the NF 魏 B signaling pathway. Therefore, RANKL-NF 魏 B-ADAMTS5 signal axis can be used as a new approach to prevent and treat articular cartilage degeneration.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782.6

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