钙卫蛋白-TLR4危险信号途径调控Th17细胞分化在种植体周围炎中的作用研究

Peri-implantitis is an inflammatory disease characterized by bone resorption, which compromise the long-term outcome of dental implants. IL-17, chiefly secreted by Th17 cells, plays an important role in inflammatory bone resorption. And Toll-like receptor 4 (TLR4) is crucial in promoting Th17 cells activation. Calprotectin is a TLR4 ligand which have a key role in the initiation and amplification of innate immune responses. However, the exact role of calprotecin in regulating Th17 cells and its role in the development of peri-implantitis is still unknown. Applicant’s previous animal study proved that calprotectin was high expressed in the peri-implant soft tissue in ligature-induced peri-implantitis. Importantly, we also found that the expression of calprotectin was significant correlated with IL-17 and bone loss. Therefore, we hypothesize that calprotectin could active Th17 cells and induce Th17 differentiation through the TLR4-signaling pathways, which promoting the development of peri-implantitis. In this study, the expression of calprotectin, TLR4 and Th17 cells in human peri-implantitis lesions would be explored. Furthermore, the role of calprotectin in active Th17 cells and induce Th17 differentiation through the TLR4-signaling pathways would be investigated. And the role of calprotectin in osteoclast precursors differentiate into osteoclasts in the presence of Th17 cells would be studied. This study would elucidate the role of calprotectin in regulating the Th17 cells and its role in the development of peri-implantitis, which would improve our understanding of the pathogenesis of peri-implantitis and provide new insights into its therapy.

种植体周围炎是以骨吸收为特征的炎性病损，影响牙种植远期疗效。Th17分泌IL-17促进炎性骨吸收并通过TLR4识别危险信号促进Th17活化。钙卫蛋白可作为TLR4配体在启动和放大固有免疫应答中起关键作用，然而，钙卫蛋白如何调控Th17细胞并在种植体周围炎中发挥作用仍未清楚。申请人前期工作证明：钙卫蛋白高表达于种植体周围炎软组织并与IL-17表达和骨吸收显著正相关。因此，我们提出科学假说：钙卫蛋白可以经TLR4途径活化并诱导Th17分化从而调节Th17的免疫学功能，促进种植体周围炎进展。本课题拟通过检测钙卫蛋白、TLR4和Th17在人种植体周围炎中的表达，分析钙卫蛋白-TLR4途径对Th17活化、分化及免疫功能的影响，研究钙卫蛋白在Th17诱导破骨前体细胞分化中的作用，初步探讨钙卫蛋白调控Th17在种植体周围炎中的作用。本项目将有助于进一步理解种植体周围炎的发病机制，为其临床治疗提供新思路。

种植体周围炎是以骨吸收为特征的炎性病损，影响牙种植远期疗效。本课题经过收集临床标本、分子生物学实验、细胞实验、基因芯片检测与生物信息学分析、动物实验结果分析，初步得到如下结果：.1. 检测了Th17、Treg及骨破坏相关炎症因子在人种植体周围软组织中的表达。为全面理解种植体周围炎中与骨破坏相关的分子，对健康牙龈组织、牙周炎及种植体周围炎牙龈组织进行基因转录谱分析，并通过qRT-PCR验证基因芯片结果并检测相关细胞因子表达水平。结果显示，与健康组相比，种植体周围炎组在细胞因子-细胞因子受体相互作用、破骨细胞分化等基因表达存在显著差异，IL-1β、IL-6、RANKL等基因mRNA表达显著上调。其中，RANKL mRNA表达水平与种植体周围探诊深度呈显著负相关。长链非编码RNA在种植体周围炎、牙周炎、牙周健康组织中表达存在显著差异。.2. 在动物实验研究中继续深入分析种植体周围炎骨吸收及软组织炎症反应的影响因素。研究结果显示，发生种植体周围炎时探诊深度增大，临床参数与骨吸收呈现正相关。组织学研究分析显示，发生种植体周围炎时种植体周围骨破坏形态受种植体-基台与骨嵴顶相对位置影响，骨水平种植体形成明显的垂直骨缺损，骨下种植体会形成更为明显的骨下袋，这可能影响疾病进展与治疗方案的选择。.3. 种植体周围炎的启动因素是菌斑生物膜的形成。本课题组成员对两种不同粗糙度的钛片表面上细菌生物膜形成进行了观察，结果显示粗糙表面的钛片促进了细菌的早期粘附，但当菌斑成熟后对细菌粘附的影响明显降低。.4. 项目组成员采用含抗菌活性的氟离子植入猪骨源性羟基磷灰石(FPHA)中，以期研发在抑菌的同时可获得良好成骨疗效的新型骨替代材料。结果显示，FPHA能有效促进成骨细胞活性。将小鼠单核巨噬细胞株RAW264.7接种在FPHA上，结果显示破骨样细胞可在FPHA上黏附、分化并发挥吸收功能，材料对前破骨样细胞的增殖活性无明显抑制作用，但可降低破骨样细胞TRAP的表达。