骨化三醇通过LL-37/miR-21/PDCD4途径促进牙龈卟啉单胞菌内化牙龈上皮细胞自噬的机制研究

Porphyromonas gingivalis, the most important periodontal putative pathogen, can escape the immune elimination by their internalization into non-phagocytic cells. Autophagy is the important way to eliminate pathogens colonizing within the cells. Our previous study proved that calcitriol decreased remarkably the numbers of Porphyromonas gingivalis in gingival epithelial cells and significantly promoted the autophagy process simultaneously, which indicates that calcitriol may promote the elimination of the intracellular Porphyromonas gingivalis by promoting the autophagy process. Our further study showed that calcitriol could promote the autophagy process by increasing the LL-37 expression, and decreasing the PDCD4 expression. We will investigate the pathway by which calcitriol induces the intracellular Porphyromonas gingivalis to be located into the mature autophagy system, and will apply CRISPR/Cas9 technique and the gene knockout mice model to explore the mechanism by which LL-37 promotes the calcitriol-inducing autophagy and by which PDCD4 inhibits the autophagy process. Also we will elucidate the roles of MAPK pathway and TGF-β standing on the crossing of LL-37 and miR-21/PDCD4 pathway. We will find out the difference between gingival epithelial cells and monocyte-macrophage. On the basis of the present project, we will learn well about the mechanism of calcitriol-mediated elimination of the intracellular Porphyromonas gingivalis and will discover the new molecular mechanism under which calcitriol regulates the autophagy process.

牙龈卟啉单胞菌是重要的牙周致病菌，可以内化非吞噬逃避免疫清除。自噬是清除细胞内寄生菌的重要方式。前期研究发现骨化三醇显著降低内化牙龈上皮细胞牙龈卟啉单胞菌数量并显著促进自噬，推测骨化三醇通过促进自噬清除细菌。随后发现骨化三醇可以通过上调LL-37表达促进自噬，并抑制PDCD4表达。本课题拟动态观察骨化三醇促进牙龈卟啉单胞菌进入自噬系统的定位途径；应用CRISPR/Cas9技术、基因敲除小鼠模型等阐明LL-37在骨化三醇诱导自噬中的正向调节作用及PDCD4的负向调节作用；探讨MAPK通路、TGF-β在LL-37和miR-21/PDCD4分子途径中的枢纽作用；比较牙龈上皮细胞和单核-巨噬细胞的异同。本课题不仅将阐明骨化三醇促进清除牙龈上皮内牙龈卟啉单胞菌的机制，而且将为骨化三醇促进自噬的分子途径研究开拓新的思路。

牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）是重要的牙周致病菌。P. gingivalis可广泛内化于口腔牙龈上皮细胞、血管内皮细胞、肝细胞、树突细胞等多种细胞，进而促进牙周炎、动脉粥样硬化、肝代谢障碍、免疫失调等多种疾病的发生和发展。本课题围绕骨化三醇抑制细胞内P. gingivalis活菌数量这一重要发现展开了系统而深入的研究，发现了重要的相关细胞生物学和分子机制。研究发现（1）在非吞噬细胞KB细胞和HaCat细胞中，P. gingivalis利用不完善的自噬过程实现在细胞内的生存和繁殖；而在吞噬细胞U937细胞中，完善的自噬过程可抑制P. gingivalis的生存。（2）骨化三醇通过促进完善的自噬过程降低细胞内P. gingivalis 的活菌数量。（3）研究充分验证了骨化三醇的靶基因LL-37通过正向促进完善自噬过程降低细胞内P. gingivalis活菌数量。（4）在 LL-37促进自噬过程中，TRIM22/LAMP3/MAPK可能是重要的分子途径。（5）大鼠牙周炎模型体内实验进一步验证了骨化三醇、LL-37在自噬和牙周炎中的调控作用。骨化三醇可促进大鼠牙周组织自噬反应和 LL-37表达，并通过促进自噬过程抑制牙周组织炎症。上述研究结果皆为首次报告的创新性成果。所获得研究成果已发表多篇中英文文章，丰富了维生素D在体内的多效性作用、P. gingivalis与宿主细胞相互作用、自噬的调控等多项理论知识体系。本研究成果将为通过调整维生素 D 水平和研制维生素 D 制剂、调控细胞内抗菌肽 LL-37水平等进而杀灭细胞内 P. gingivalis，从而预防和治疗牙周炎及多种系统性疾病的应用研究提供充足可靠的科学依据。未来将实现成果转化，争取获得更大的社会效益和经济效益。