本文選題：牙齦卟啉單胞菌　切入點：壞死性凋亡　出處：《南京大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]Necroptosis是一種炎性的細胞死亡方式,可產(chǎn)生大量損傷相關(guān)分子型(damage-associatedmolecularpatterns,DAMPs)。它包含三個關(guān)鍵蛋白,受體相互作用蛋白激酶-1(receptor interacting protein kinase,RIP1),RIP3 及其底物混合系列蛋白激酶樣結(jié)構(gòu)域(mixed lineage kinase like,MLKL)。本實驗的研究目的是探討necroptosis是否影響牙齦卟啉單胞菌(Porphyromonasgingivalis)引起的炎癥免疫應(yīng)答和牙周炎的發(fā)病過程。[方法]在體外實驗中,對THP-1細胞給予P.gingivalis刺激,Western Blot檢測necroptosis的分子標記物-磷酸化MLKL的表達情況,確認necroptosis的發(fā)生;采用RIP1和MLKL的抑制劑Nec-1和NSA處理THP-1細胞,ELISA檢測細胞在P.gingivalis刺激條件下分泌炎癥因子水平的變化,LDH檢測細胞死亡情況,分析necroptosis在應(yīng)答P.gingivalis反應(yīng)過程中的炎癥作用:使用慢病毒干擾序列siRNA分別沉默RIP3和MLKL,進一步明確necroptosis影響單核細胞應(yīng)答P.gingivalts的急性炎癥反應(yīng)。為進一步研究necroptosis在急性炎癥中的作用,采用小鼠皮下小室模型,在小室內(nèi)注射P.gingivalis造成急性感染,于不同時間點抽取小室滲出液,進行P.gingivalis培養(yǎng)和炎癥因子檢測,觀察necroptosis對體內(nèi)細胞清除P.gingivalis和釋放炎癥因子水平的影響。為進一步研究necroptosis在慢性炎癥中的作用,在小鼠牙周炎模型實驗中,用P.gingivalis提前浸泡過的絲線對小鼠雙側(cè)上頜第二磨牙進行結(jié)扎,1天后取出結(jié)扎絲線。每日給予NSA腹腔注射,隔天給予P.gingivalis齦溝內(nèi)注射,持續(xù)2周,觀察necroptosis對牙周炎發(fā)病過程的影響。[結(jié)果]THP-1細胞經(jīng)P.gingivalis刺激8 h后檢測到了磷酸化MLKL的表達。特異性抑制劑Nec-1和NSA均明顯下調(diào)了 THP-1細胞分泌TNF-a和IL-6的水平;THP-1細胞使用NSA處理后,或者慢病毒siRNA沉默RIP3和MLKL后,P.gingivalis導(dǎo)致的細胞死亡顯著減少。皮下小室感染1d后,NSA處理組的滲出液P.gingivalis的數(shù)量明顯低于對照組。在1h和8h,NSA處理顯著下調(diào)了小室滲出液中的TNF-α水平;而IL-6的表達在8h和1d也明顯受到抑制。在小鼠牙周炎模型中,第二磨牙絲線結(jié)扎+齦溝內(nèi)注射P.gingivalis引起明顯的炎癥性骨吸收,而NSA處理組的牙槽骨吸收量明顯減少。并且,在P.gingivalis組的牙周組織中,磷酸化MLKL的免疫組化染色呈陽性。[結(jié)論]P.gingivalis可以激活THP-1細胞發(fā)生RIP3/MLKL介導(dǎo)的necroptosis,抑制necroptosis死亡通路能夠有效減少P.gingivali 產(chǎn)生的細胞死亡,下調(diào)炎癥因子分泌水平;在急性感染小室中阻斷necroptosis可以減少炎癥滲出水平,并有利于P.gingivalis清除;necroptosis參與了P.gingivalis誘導(dǎo)的牙周炎過程,阻斷necroptosis可以減少P.gingivalis導(dǎo)致的炎癥性牙槽骨吸收,延緩牙周炎進展。
[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- 偽 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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.42
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本文編號：1591720