Salubrinal在人髓核细胞中抑制IL-17介导的椎间盘退变机制的研究

发布时间：2018-03-19 11:42

本文选题：白介素17　切入点：椎间盘退变　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景腰椎间盘退变疾病是骨科临床常见病,其主要特征是细胞外基质(ECM)成分下降,水分减少,胶原比例变化等。基质金属蛋白酶(MMPs)在这一过程中扮演重要角色。MMPs一般通过中性粒细胞、巨噬细胞、纤维母细胞等组织细胞产生,是一类Ca和Zn依赖的蛋白水解酶家族,参与降解全身各种组织细胞外基质成分。有研究表明,基质金属蛋白酶13(MMP-13)主要来源于软骨细胞,具有降解Ⅱ型胶原和聚集蛋白聚糖(Aggrecan)的作用,因此,基质金属蛋白酶13在基质降解过程里可以发挥双重作用。以往的研究表明,MMP-13在炎性介质(白介素-1β(IL-1β)、肿瘤坏死因子α(TNF-α)等)介导的人椎间盘退变过程中起重要作用。但是关于白介素-17(IL-17)与MMP-13在椎间盘退变过程中的关系尚未有相关报道。Salubrinal是真核起始因子2α(eIF2α)去磷酸化的抑制剂,能够提升eIF2α的磷酸化水平,从而抑制内质网应激引起的细胞凋亡。最近的研究表明,salubrinal具有促进骨愈合、抑制骨质疏松、抑制骨细胞和成骨细凋亡及抑制骨关节炎等骨保护作用,但是关于salubrinal与椎间盘退变的关系尚未有人报道,本实验将研究salubrinal是否可抑制IL-17介导的椎间盘退变过程。研究目的1.本实验将研究IL-17在椎间盘退变过程中对基质金属蛋白酶-13和细胞外基质成分的调节。2.研究真核起始因子2α去磷酸化的抑制剂salubrinal,是否可以抑制IL-17介导的上述过程。研究方法1.选取2014年2月-2014年9月在山东大学齐鲁医院骨外科住院住院并经临床表现和影像学检查确诊为腰椎间盘突出症患者。20个取自经椎间孔融合术及后路腰椎椎间融合术患者的椎间盘,所有椎间盘按Pfirrmann评分属于非退变组,椎间盘分离髓核组织后,提取原代髓核细胞并传代培养。2.用蛋白免疫印迹和RT-PCR技术,检测不同浓度和不同时间IL-17刺激下,髓核细胞中MMP-13、Ⅱ型胶原(COL2A1)、Ⅰ型胶原(COL1A1)及聚集蛋白多糖(ACAN)的表达。3.用蛋白免疫印迹和免疫荧光技术,检测IL-17刺激下,NF-κB通路是否被激活。另外,用蛋白免疫印迹技术,检测IL-17或salubrinal刺激下,eIF2α的表达情况。4.用蛋白免疫印迹和RT-PCR技术,检测在salubirnal或NF-κB通路的抑制剂与IL-17共同刺激下,髓核细胞中MMP-13、Ⅱ型胶原、Ⅰ型胶原和聚集蛋白多糖的表达,以及NF-κB通路是否被激活。研究结果1.蛋白免疫印迹实验表明,IL-17刺激髓核细胞,使MMP-13和Ⅰ型胶原表达升高,并降低Ⅱ型胶原和聚集蛋白多糖的表达,RT-PCR实验表明,IL-17刺激髓核细胞,使MMP-13表达升高,并降低Ⅱ型胶原、Ⅰ型胶原和聚集蛋白多糖的表达。2.免疫印迹实验及免疫荧光实验表明,IL-17刺激髓核细胞激活了 NF-κB通路。另外,免疫印迹实验表明,IL-17同时使eIF2α的表达升高。进一步的实验表明,在IL-17和BAY11-7082共同刺激下,eIF2α磷酸化水平并没有显著性差异(P0.05)。3.Salubrinal和NF-κB通路抑制剂BAY11-7082可以抑制IL-17激活的NF-κB通路。同时,免疫印迹技术表明,salubrinal和BAY11-7082抑制了 IL-17导致的MMP-13和Ⅰ型胶原表达升高,以及Ⅱ型胶原、聚集蛋白多糖表达下降。RT-PCR表明,salubrinal 和 BAY 11-7082 抑制了 IL-17 导致的 MMP-13 表达升高,以及Ⅱ型胶原、Ⅰ型胶原、聚集蛋白多糖表达下降。结论IL-17在人髓核细胞中通过激活NF-κB通路提高了 MMP-13的表达同时抑制COL2A1和ACAN的表达;salubrinal可以抑制IL-17介导的椎间盘退变过程,从而为salubrinal治疗椎间盘退变提供了依据。
[Abstract]:The research background of disease of lumbar disc degeneration is a common disease in clinical department of orthopedics, its main feature is the extracellular matrix (ECM) components decreased, moisture decreased, collagen proportion changes. Matrix metalloproteinases (MMPs) play an important role in.MMPs by granulocyte macrophage, in this process, fibroblast cells, is a family of proteolytic enzymes like Ca and Zn dependent, all organizations involved in the degradation of extracellular matrix components. Studies have shown that matrix metalloproteinase 13 (MMP-13) mainly derived from chondrocytes, with degradation of type II collagen and aggrecan (Aggrecan) role, therefore, matrix metalloproteinase 13 in the substrate degradation process can play a dual role. Previous studies showed that MMP-13 in inflammatory mediators (interleukin -1 beta (IL-1 beta), tumor necrosis factor alpha (TNF- alpha) etc.) degeneration of the human intervertebral disc mediated in An important role. But on interleukin -17 (IL-17) and MMP-13 in the process of disc degeneration has not been reported that.Salubrinal is the eukaryotic initiation factor 2 alpha (eIF2 alpha) inhibitor to phosphorylation, can enhance the phosphorylation level of eIF2 alpha, thereby inhibiting the apoptosis induced by endoplasmic reticulum stress in recent. Research shows that salubrinal can promote bone healing, inhibiting osteoporosis, inhibition of bone cells and osteoblasts apoptosis and inhibit bone arthritis and other bone protective effect, but the relationship between salubrinal and intervertebral disc degeneration has not yet been reported, this experiment will investigate whether salubrinal can inhibit IL-17 mediated degeneration of the intervertebral disc. The purpose of the study 1. the experiment study of IL-17 in the process of disc degeneration on matrix metalloproteinase -13 and extracellular matrix components regulating.2. of eukaryotic initiation factor 2 alpha phosphorylation inhibitor salubrinal, is The process can not mediated by inhibition of IL-17. Methods: 1. from February 2014 -2014 year in September in the Qilu Hospital of Shandong University hospital and bone surgery by clinical and radiological examination confirmed the diagnosis of lumbar disc herniation patients with.20 from the disc intervertebral fusion and posterior lumbar interbody fusion of lumbar intervertebral disc patients, all according to the Pfirrmann score belong to non degeneration group, intervertebral disc nucleus pulposus after separation, extraction of primary nucleus pulposus cells and Western blotting and RT-PCR technology with the.2. subculture, detection of different concentration and time of IL-17 under the stimulation of MMP-13 in the nucleus pulposus cells, collagen type II (COL2A1), collagen (COL1A1) and protein aggregation sugar (ACAN) protein expression by Western blotting and immunofluorescence technique with.3. detection, IL-17 stimulation, NF- B pathway is activated. In addition, by Western blot technique, the detection of IL-17 or salubrina Under the stimulation of L eIF2 alpha.4. expression by Western blotting and RT-PCR detection technology, in the salubirnal or NF- inhibitor and IL-17 B pathway together under the stimulation of MMP-13 in the nucleus pulposus cells, the expression of type II collagen, type I collagen and aggrecan, and the NF- B pathway is activated 1.. The results of Western blot showed that IL-17 stimulation of nucleus pulposus cells, MMP-13 and collagen expression increased, and decreased the expression of type II collagen and aggrecan, RT-PCR results showed that IL-17 stimulation of nucleus pulposus cells, increase the expression of MMP-13 and decrease that of type II collagen, type I collagen and the aggrecan.2. expression by Western blotting and immunofluorescence experiments, IL-17 stimulated the activation of NF- B pathway in nucleus pulposus cells. In addition, immunoblotting experiments showed that IL-17 and eIF2 expression increased. Further experiments showed that, in IL-17 and BAY11-708 2 common stimulation, there is no significant difference between the phosphorylation level of eIF2 alpha (P0.05) NF- BAY11-7082.3.Salubrinal NF- B pathway and B pathway inhibitors can inhibit the activation of IL-17. At the same time, that Western blot, salubrinal and BAY11-7082 inhibited MMP-13 and IL-17 induced expression of collagen type I and type II collagen, increased. Aggrecan decreased the expression of.RT-PCR showed that salubrinal and BAY 11-7082 inhibited IL-17 caused increased expression of MMP-13 and collagen type II collagen and aggrecan expression decreased. Conclusion IL-17 in human nucleus pulposus cells by expression of increased MMP-13 expression and inhibit COL2A1 and ACAN activation of NF- B pathway. Intervertebral disc degeneration; salubrinal can inhibit IL-17 mediated by salubrinal, which provides a basis for the treatment of intervertebral disc 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】：R681.53

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