MicroRNA-155对牙龈卟啉单胞菌OmpH诱导巨噬细胞Pyroptosis调控机制研究

Inflammasome activation in macrophages of human periodontal tissue results in a form of cell death termed 'pyroptosis' , on the one hand it will reduce the ability to clear pathogens in periodontal tissue, so that periodontal disease is difficult to control. On the other hand , due to the consequent release of intracellular contents, the extent of periodontal inflammatory reaction is increased, and absorption of tooth-supporting alveolar bone is promoted . In this study, we will construct ompH (PG0192 and PG0193) gene knockout strains and revertant strains of P.gingivalis W83. Macrophages U937 and macrophages from human peripheral blood will be stimulated with P.gingivalis W83 ompH gene knockout strains and revertant strains.We construct OmpH protein expression vector and the macrophages are stimulated by purified proteins. We will use several methodes to evaluate the effection of OmpH protein on macrophage miR-155 expression and cell pyroptosis. Analyse the mechanisms that miR-155 regulate cell pyroptosis through TLR4-TRIF-IFN-Caspase-11 signaling pathway. Through animal experiments, we will confirm the correctness of the mechanisms about miR-155 in macrophage pyroptosis. We are going to find a way to suppress pyroptosis when macrophages are stimulated with P.gingivalis , enhance the ability of periodontal tissue resistance to infection and reduce the possibility of periodontal infection aggravation and recurrence.

人牙周组织中的巨噬细胞发生Pyroptosis，一方面会降低牙周组织清除致病菌的能力，使牙周炎症不易控制，另一方面由于胞内物质流出，加重了牙周炎症反应程度，促进了牙槽骨的吸收。因此本研究拟通过构建 ompH（PG0192 和PG0193）基因敲除株和ompH回复突变菌株，用P.gingivalis W83野生株及ΔompH及回复突变菌株感染人巨噬细胞U937和人外周血巨噬细胞，同时构建 OmpH蛋白表达载体，明确P.gingivalis 外膜蛋白OmpH对巨噬细胞miR-155的表达及Pyroptosis的影响，确定miR-155对TLR4-TRIF-IFN- Caspase-11途径的调控机制，并通过动物实验验证机制的正确性。希望通过此项研究能够发现一条途径阻断P.gingivalis感染后巨噬细胞的Pyroptosis，增强牙周组织抗感染的能力，减轻牙周炎症和牙周病复发的可能性。

牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）是重要的牙周致病菌，巨噬细胞在牙周组织局部发挥吞噬功能，并释放各种促炎因子，促进牙槽骨吸收及牙周组织破坏。巨噬细胞发生Pyroptosis，一方面会降低清除致病菌的能力，使牙周炎症不易控制，另一方面由于胞内物质流出，加重了牙周炎症反应，促进牙槽骨的吸收。.本课题组体外建立了P. gingivalis W83感染人U937巨噬细胞模型，从形态学观察pyroptpsis的发生，并且IL-1β、IL-18表达上升，检测CAS-11 、NLRP3复合体 mRNA表达增高，通过Western blot法检测炎性相关蛋白表达升高，发现用P. gingivalis刺激后，巨噬细胞Pyroptosis率和miR-155的表达显著增加，当miR-155沉默时Pyroptosis发生率降低；发现miR-155通过调节NLRP3炎性体参与P. gingivalis促进巨噬细胞Pyroptosis的过程；其次，我们制备重组蛋白rOMPH-1、rOMPH-2，明确OmpH1和OmpH2的加入可以使P. gingivalis W83诱导巨噬细胞IL-6 和TNF-α mRNA合成增多，该作用可能与巨噬细胞表面的C5aR受体有关。.之后我们电转构建P. gingivalis W83突变株，用P. gingivalis W83野生株、突变株△PG0192-0193和回复株com△PG0192-0193分别诱导巨噬细胞株U937，结果显示其对巨噬细胞U937的受体TLR2表达量及细胞分泌TNF-α、IL-1β、IL-6影响能力不同；接下来我们以miScript miRNA PCR Array来分析△PG0192-0193和 P. gingivalis W83感染巨噬细胞后miRNA表达谱的差异，发现表达上调的有15个miRNA，表达下调的有10个miRNA，并通过Targetscan等数据库预测相应miRNA的靶基因，结果说明外膜蛋白OmpH可能通过感染巨噬细胞后miRNA表达的变化来调节炎症反应；由于巨噬细胞Pyroptosis的发生加重了牙周炎症反应及牙槽骨的吸收，因此我们又检测了P. gingivalis刺激巨噬细胞来源的外泌体对成骨前体细胞的影响，发现该外泌体能抑制成骨前体细胞增殖，促进其凋亡。