应用基因敲除小鼠探讨组织蛋白酶S在慢性牙周炎进程中的作用及分子机理

Chronic periodontitis(CP) is an inflammatory condition of the periodontium, which is the primary cause of tooth loss and is also a risk factor for the systemic health in adults worldwide. The etiology of periodontitis is recognized to be multifactorial, however, precise mechanisms are still need to clarify. In recent years, increasing evidences indicated that host immune mechanism plays an essential role in CP. Antigen antibody complexes of oral bacteria trigger antigen-presenting cells(APC) reaction including dentritic cells and macrophages, leading to initiate immune response. As a result of cellular activation, inflammatory mediators contribute to tissue destruction and bone resorption. Cathepsin S (CatS), a lysosomal cysteine protease preferentially expressed in mononuclear-phagocytic cells, is responsible for the final proteolytic cleavage stage of Ii from lip10 to CLIP in APC. Our previous study found that CatS deficient mice could suppress CP through inhibiting expressions of immune cells and pro-inflammatory cytokines in inflammatory periodontal tissues. We also confirmed that the up-regulation of CatS expression in APC stimulated by Porphyromonas gingivalis is related to antigen presentation. Based on these results, firstly, we will clarify the effect of CatS deficiency to the localization of immune cells and immunity cytokines in gingiva tissues of CP mice model using immunohistochemical technologies. Secondly, we will determine the influence of CatS deficiency to the APC antigen-presenting function by flow cytometry and proteomic technologies such as qPCR, western blot as well as ELISA in vitro. Finally, we will compare the serological indicators between wild type and CatS deficient CP mice to elucidate the molecular mechanism of CatS in the pathogenesis of CP. We believe that our projects will give a new viewpoint for the therapeutic option of CP.

慢性牙周炎是由多种细菌作为始动因子的慢性炎症性疾病，但其发生及慢性化机制还尚未明确。组织蛋白酶S(CatS)主要表达于抗原提呈细胞，通过对lip10至CLIP的降解在抗原提呈中起关键作用。我们在前期研究中首次发现CatS基因缺失小鼠通过抑制炎性细胞及促炎因子在炎症牙龈中的表达而抑制小鼠牙周炎的发病；利用单细胞分离机联合免疫磁珠技术提取抗原提呈细胞，发现在牙龈卟啉单胞菌激活下抗原提呈细胞中CatS高表达与抗原提呈相关。本项目拟利用CatS基因缺失小鼠作为研究动物模型，通过采用免疫染色等方法明确CatS基因缺失对慢性牙周炎小鼠牙龈组织内免疫细胞的分布及炎性因子产生的影响；流式细胞法、Western blot等细胞及分子生物学方法明确CatS基因缺失对抗原提呈细胞的抗原提呈功能及对免疫细胞亚群分化的影响，阐明CatS在慢性牙周炎进程中的作用及分子机理，为临床上慢性牙周炎的靶向治疗提供一条新途径。

脑内的小胶质细胞特异性表达CatS，并且是由小胶质细胞生物钟调控的。我们发现在CatS基因缺损小鼠大脑皮层中神经元诱发突触反应的日变化消失。大脑皮层神经元注入CatS重组蛋白后可观察到其树突棘密度显著降低。此外，我们通过three-chamber实验发现CatS-/-小鼠在社交以及社会新奇性认知方面受损。以上这些发现明确指出由小胶质细胞生物钟调控的小胶质细胞分泌的CatS通过分解树突棘周围细胞外基质参与了调节大脑皮层中神经元诱发突触反应的日变化与树突棘密度变化。因此CatS基因缺失可能会引起社会行为异常。.中药防已黄芪汤（TJ-20）是由六种草药配制而成的，在中国与日本应用于类风湿性关节炎的临床治疗（RA）已经有很长的历史。但是其科学根据还尚未明确。在现阶段的研究中，我们专注于探讨TJ-20对使用费氏完全佐剂（CFA）诱导的大鼠关节炎模型的主要作用和治疗效果。我们分别使CFA诱导的关节炎大鼠从关节炎第0天，第7天，第10天开始口服TJ-20，服用至8周， 每日服用量600mg/kg。TJ-20随着服用时间而显著改善关节炎大鼠的炎症进程及骨破坏。此外，TJ-20显著抑制巨噬细胞以及辅助性T细胞的增长。而且TJ-20可抑制TNF-a但促进IL-10分泌，并且使Th1细胞应答减弱。因此，TJ-20在关节炎大鼠模型中可调控巨噬细胞促炎因子抗炎因子的表达及Th1/Th2的平衡。以上结论显示TJ-20的抗炎效果并且为其在RA的临床上提供了科学依据。