【作者】 刘杰；

【导师】 王敏；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 硕士

【摘要】 根尖周炎是口腔最常见的感染性疾病之一,由牙髓病原体引起根尖区炎症和根周组织破坏。根尖周炎的病程进展和转归取决于根管微生物与宿主防御之间的动态平衡。当致病因素能被消除时,根尖炎症不会引起骨质破坏和吸收;但当致病因素不能被消除时,则会介导根尖区骨质吸收和组织破坏。虽然已有较多关于根尖周炎发生发展的研究,但参与其中的多种分子机制尚未研究透彻。近年来,一种由受体相互作用蛋白激酶1(receptor interacting protein kinase-1,RIP1)、受体相互作用蛋白激酶3(receptor interacting protein kinase-3,RIP3)和混合系谱激酶结构域样蛋白mixed lineage kinase like,MLKL)介导的细胞死亡方式,即程序性细胞坏死被广泛研究。它是近年来新发现的一种细胞调控性死亡,该方式致使细胞破坏是通过形成死亡小体。程序性细胞坏死参与了许多炎症疾病如回肠炎、皮肤炎症、系统性炎症反应综合症的发生发展,能被肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、细菌脂多糖(lipopolysaccharide,LPS)、细胞膜上的Toll样受体(Toll-like receptors,TLRs)等分子介导激活,而上述分子也与根尖周炎的发生发展密切相关,这提示了二者之间的潜在联系。然而根尖周炎的发生发展是否有程序性细胞坏死的参与,并未见文献报道。目的:通过体内体外实验,探索程序性细胞坏死在根尖周炎发生发展中的作用,从而揭示根尖周炎潜在的致病机制,以期为根尖周炎的防治提供新的策略。方法:1.利用髓腔定植根尖周炎致病菌--具核梭杆菌(Fusobacterium nucleatum,Fn)的方法构建根尖周炎小鼠疾病模型。通过微计算机断层扫描(Micro-computed tomography,Micro-CT)、苏木精-伊红染色(Hematoxylin-eosin,HE)以及破骨细胞抗酒石酸酸性磷酸酶染色(Enzyme activity staining of tartrateresistant acid phosphatase,TRAP)对根尖周病损区进行硬组织形态学分析和骨吸收进行评估。利用免疫组化(Immunohistochemistry,IHC)染色检测程序性细胞坏死通路中关键分子RIP3和磷酸化MLKL(Phosphorylated mixed lineage kinase like,p MLKL)的表达情况。2.体外培养L929小鼠成纤维细胞系,通过Fn模拟炎症刺激,并给予TNF-α、凋亡抑制蛋白抑制剂-AZD5582和酪蛋白酶抑制剂-z VAD-FMK共同处理诱导L929程序性细胞坏死,采用蛋白免疫印迹法(Western Blotting,WB)、免疫荧光(Immunofluorescence,IF)、定量逆转录聚合酶链式反应(Quantitative RealTime PCR,q RT-PCR)和细胞坏死活力检测试验,检测Fn对L929的刺激作用。3.构建根尖周炎小鼠疾病模型,使用腺病毒相关病毒(Adenovirus associated virus,AAV)抑制程序性细胞坏死关键分子RIP3的表达。成纤维细胞系L929和Fn共培养,小干扰RNA(Small interfering RNA;si RNA)转染抑制RIP3的表达。通过Micro-CT及TRAP染色对根尖周病损区进行硬组织形态学分析,对各组骨吸收进行评估。通过WB、IHC染色及q RT-PCR对各组根尖周区域的RIP3和p MLKL的表达和下游炎性细胞因子进行检测,探究根尖周炎中程序性细胞坏死的作用。结果:1.根尖周炎疾病模型中,骨吸收和破骨细胞数量随时间而增加,程序性细胞坏死通路关键分子p MLKL表达升高。2.在成纤维细胞中,Fn刺激可以上调程序性细胞坏死通路中关键分子RIP3、p RIP3、MLKL、p MLKL的表达和炎症基因m RNA表达水平,并可以加剧细胞坏死降低细胞活性。3.在成纤维细胞中抑制RIP3后,p MLKL表达降低,细胞活力增强,坏死减少,相关炎症因子基因的m RNA表达水平降低;根尖周炎疾病模型中,抑制RIP3蛋白表达后,p MLKL表达降低,破骨细胞数量减少,根尖区骨质吸收减少,炎症基因m RNA表达水平降低。结论:在受Fn刺激的L929细胞和小鼠根尖周炎模型中,程序性细胞坏死通路被激活,炎症表型加剧;抑制程序性细胞坏死通路中的RIP3之后,其关键执行分子p MLKL表达水平降低,破骨细胞数量和相关炎症因子基因m RNA表达水平降低,小鼠根尖周炎骨吸收减少,表明程序性细胞坏死参与了根尖周炎的致病过程。更多还原

【Abstract】 Apical periodontitis is one of the most common infectious diseases of the oral cavity.The inflammation of the apical area and destruction of the periapical tissues are caused by dental pulp pathogens.The course and outcome of apical periodontitis depends on the dynamic balance between root canal microorganisms and host defense.When the pathogenic factors can be eliminated,the inflammation of the root tip will not cause bone destruction and resorption;but when the pathogenic factors cannot be eliminated,it will mediate bone resorption and tissue destruction in the root tip area.Although there have been many studies on the occurrence and development of periapical periodontitis,the various molecular mechanisms involved in it have not been thoroughly studied.In recent years,necroptosis,a newly found cell-regulated death which is induced by receptor interacting protein kinase-1(RIP1),receptor interacting protein kinase-3(RIP3)and mixed lineage kinase like(MLKL),has been studied wildly.Necroptosis can trigger cell damage via formation of necrosome.Studies have shown that necroptosis is involved in various inflammatory diseases such as enteritis,skin inflammation,and systemic inflammatory response syndrome.Programmed cell necrosis can be activated by molecules such as lipopolysaccharide(LPS),tumor necrosis factor α(TNF-α),Toll-like receptors(TLRs),etc.,and these molecules also play an important role in the occurrence and development of apical periodontitis,suggesting a potential correlation between the necroptosis and apical periodontitis.However,whether necroptosis is involved in apical periodontitis has not been reported in the literature.Objective: To explore the role of necroptosis in the progress of apical periodontitis through in vivo and in vitro experiments,so as to reveal the possible new pathogenic mechanism of apical periodontitis,and provide new strategies for the prevention and treatment of apical periodontitis.Methods: 1.Fterium nucleatum(Fn)was colonized in the pulp cavity to construct a disease model of apical periodontitis in mice.Micro-computed tomography(Micro-CT)and osteoclast enzyme activity staining of tartrate-resistant acid phosphatase(TRAP)were used to analyze bone resorption in each group.The expression of RIP3 and p MLKL,the key molecules of necroptosis were detected by WB and immunohistochemistry(IHC).2.Mouse fibroblast cell line L929 were co-cultured with Fn or received cotreatment of TNF-α,AZD5582 and z VAD-FMK to induce necroptosis.Protein Western blotting(WB),immunofluorescence(IF)、quantitative reverse transcription polymerase chain reaction(q RT-PCR)and cell death and viability test were used to detect the effects of Fn on L929.3.A mouse disease model of apical periodontitis was constructed,where adenovirus associated virus(AAV)was used to inhibit the expression of RIP3.The fibroblast cell line L929 and Fn were co-cultured,and small interfering RNA(si RNA)was transfected into L929 cells to inhibit the expression of RIP3.Micro-CT and TRAP staining of osteoclasts were used to analyze bone resorption in each group.WB,IHC staining and q RT-PCR were used to detect the expression of RIP3 and p MLKL and downstream inflammatory cytokines in the periapical region.Results: 1.In the mice model of apical periodontitis,expression of RIP3 and p MLKL was enhanced compared to the control group.2.Infection of Fn up-regulated the expression of RIP3,p RIP3,MLKL and p MLKL.Death of L929 cells was promoted by infection of Fn while viability of L929 was decreased.3.After the inhibition of RIP3 in the L929 cells,expressions of p MLKL and m RNA of inflammatory cytokines were decreased while cell viability was promoted.Apical lesions suffered ameliorated bone resorption after the inhibition of RIP3,and decreased number of osteoclasts,decreased expression of p MLKL and downregulated m RNA of inflammatory cytokines were noted.Conclusion: Infection of Fn can trigger necroptosis in L929 cells and apical periodontitis of mouse and aggravate the inflammatory phenotype;inhibiting RIP3 in the necroptosis pathway decreases the expression of the key executive molecule p MLKL and m RNA of inflammatory cytokines,thus alleviate the bone resorption in the apical lesion.In summary,necroptosis is involved in the progress of apical periodontitis,and the discovery of the study may provide a new target for the prevention and treatment of apical periodontitis.更多还原