RIP3介导的坏死性凋亡在牙周炎发病过程中的作用

发布时间：2018-03-10 04:16

  本文选题：牙龈卟啉单胞菌　切入点：坏死性凋亡　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]Necroptosis是一种炎性的细胞死亡方式,可产生大量损伤相关分子型(damage-associatedmolecularpatterns,DAMPs)。它包含三个关键蛋白,受体相互作用蛋白激酶-1(receptor interacting protein kinase,RIP1),RIP3 及其底物混合系列蛋白激酶样结构域(mixed lineage kinase like,MLKL)。本实验的研究目的是探讨necroptosis是否影响牙龈卟啉单胞菌(Porphyromonasgingivalis)引起的炎症免疫应答和牙周炎的发病过程。[方法]在体外实验中,对THP-1细胞给予P.gingivalis刺激,Western Blot检测necroptosis的分子标记物-磷酸化MLKL的表达情况,确认necroptosis的发生;采用RIP1和MLKL的抑制剂Nec-1和NSA处理THP-1细胞,ELISA检测细胞在P.gingivalis刺激条件下分泌炎症因子水平的变化,LDH检测细胞死亡情况,分析necroptosis在应答P.gingivalis反应过程中的炎症作用:使用慢病毒干扰序列siRNA分别沉默RIP3和MLKL,进一步明确necroptosis影响单核细胞应答P.gingivalts的急性炎症反应。为进一步研究necroptosis在急性炎症中的作用,采用小鼠皮下小室模型,在小室内注射P.gingivalis造成急性感染,于不同时间点抽取小室渗出液,进行P.gingivalis培养和炎症因子检测,观察necroptosis对体内细胞清除P.gingivalis和释放炎症因子水平的影响。为进一步研究necroptosis在慢性炎症中的作用,在小鼠牙周炎模型实验中,用P.gingivalis提前浸泡过的丝线对小鼠双侧上颌第二磨牙进行结扎,1天后取出结扎丝线。每日给予NSA腹腔注射,隔天给予P.gingivalis龈沟内注射,持续2周,观察necroptosis对牙周炎发病过程的影响。[结果]THP-1细胞经P.gingivalis刺激8 h后检测到了磷酸化MLKL的表达。特异性抑制剂Nec-1和NSA均明显下调了 THP-1细胞分泌TNF-a和IL-6的水平;THP-1细胞使用NSA处理后,或者慢病毒siRNA沉默RIP3和MLKL后,P.gingivalis导致的细胞死亡显著减少。皮下小室感染1d后,NSA处理组的渗出液P.gingivalis的数量明显低于对照组。在1h和8h,NSA处理显著下调了小室渗出液中的TNF-α水平;而IL-6的表达在8h和1d也明显受到抑制。在小鼠牙周炎模型中,第二磨牙丝线结扎+龈沟内注射P.gingivalis引起明显的炎症性骨吸收,而NSA处理组的牙槽骨吸收量明显减少。并且,在P.gingivalis组的牙周组织中,磷酸化MLKL的免疫组化染色呈阳性。[结论]P.gingivalis可以激活THP-1细胞发生RIP3/MLKL介导的necroptosis,抑制necroptosis死亡通路能够有效减少P.gingivali 产生的细胞死亡,下调炎症因子分泌水平;在急性感染小室中阻断necroptosis可以减少炎症渗出水平,并有利于P.gingivalis清除;necroptosis参与了P.gingivalis诱导的牙周炎过程,阻断necroptosis可以减少P.gingivalis导致的炎症性牙槽骨吸收,延缓牙周炎进展。
[Abstract]:[objective] Necroptosis is an inflammatory cell death mode that produces a large number of damage-associated molecular types of damage-associated molecular patterns.It contains three key proteins. The purpose of this study was to investigate whether necroptosis affects the inflammatory immune response induced by Porphyromonas gingivalis and periodontal disease. The pathogenesis of inflammation. [methods] in vitro experiments, THP-1 cells were stimulated by P.gingivalis. The expression of phosphorylated MLKL, a molecular marker of necroptosis, was detected by Western Blot to confirm the occurrence of necroptosis. RIP1 and MLKL inhibitor Nec-1 and NSA treated THP-1 cells were used to detect the level of inflammatory cytokines secreted by THP-1 cells stimulated by P.gingivalis. To analyze the inflammatory effect of necroptosis in response to P.gingivalis reaction: RIP3 and MLKL were silenced by lentivirus interference sequence siRNA, and the acute inflammatory response of monocytes to P.gingivalts was further clarified by necroptosis. In order to further study the role of necroptosis in acute inflammation, Acute infection was induced by injecting P.gingivalis into mouse subcutaneous compartment model. The exudates were extracted at different time points for P.gingivalis culture and inflammatory factor detection. To observe the effect of necroptosis on the clearance of P.gingivalis and the release of inflammatory cytokines in vivo, in order to further study the role of necroptosis in chronic inflammation, in mouse periodontitis model experiment, Bilateral maxillary second molars were ligated with P. gingivalis filaments in advance for 1 day. NSA was given intraperitoneally daily and P.gingivalis was injected into gingival sulcus the next day for 2 weeks. To observe the effect of necroptosis on the pathogenesis of periodontitis. [results] the expression of phosphorylated MLKL was detected in THP-1 cells stimulated by P.gingivalis for 8 h. The specific inhibitor Nec-1 and NSA significantly decreased the level of TNF-a and IL-6 secreted by THP-1 cells. Or the cell death induced by lentivirus siRNA silencing RIP3 and MLKL was significantly decreased. The quantity of exudate P.gingivalis in the treatment group was significantly lower than that in the control group after 1 day of subcutaneous ventricular infection. At 1 h and 8 h, the level of TNF- 伪 in the exudate was significantly down-regulated. The expression of IL-6 was also inhibited at 8h and 1d. In the mouse model of periodontitis, injection of P. gingivalis into the gingival sulcus by ligation of the second molar filaments led to obvious inflammatory bone resorption, while the alveolar bone resorption was significantly decreased in the NSA treatment group. In the periodontal tissues of P.gingivalis group, the expression of phosphorylated MLKL was positive by immunohistochemistry. [conclusion] P.gingivalis can activate THP-1 cells to induce RIP3/MLKL mediated necrotosis.The inhibition of necroptosis death pathway can effectively reduce the cell death produced by P. gingivali and down-regulate the level of inflammatory factor secretion. Blocking necroptosis in the acute infection chamber can reduce the level of inflammatory exudation and help P. gingivalis eliminate necropism participate in the process of periodontitis induced by P. gingivalis. Blocking necroptosis can reduce the inflammatory alveolar bone resorption induced by P. gingivalis and delay the progress of periodontitis.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.42
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本文编号：1591720