巨噬细胞移动抑制因子在牙龈卟啉单胞菌增强血管内皮细胞黏附活性中的作用机制研究

Porphyromonas gingivalis is a major pathogen in chronic periodontitis. Both epidemiologic and experimental findings suggest that infection with P. gingivalis exacerbates progression of atherosclerosis. Macrophage migration inhibitory factor (MIF) is a pleotropic cytokine that promotes atherogenic leukocyte recruitment via intercellular adhesion molecule (ICAM)-1 and the contribution of MIF to atherosclerotic vascular disease has come into focus of many studies in recent years. Our study had shown that cell surface expression of ICAM-1 is increased in vascular endothelial cells challenged by P. gingivalis; The adhesion of monocytes to endothelium is enhanced by P. gingivalis as well as the expression of MIF. Namely, the adhesive properties of endothelial cells are increased by pretreatment of P. gingivalis. These observations raise the possibility that MIF affected the adhesive properties of endothelial cells challenged by P.gingivalis. Therefore, the first aim of this study was to examine the ability of MIF to regulate endothelial ICAM-1 expression and monocyte–endothelial cell interactions with infection of P. gingivalis. An additional poorly understood aspect of the MIF-dependent effect on adhesive properties is the identity of the cellular receptor via which MIF mediates its effect. Then the role of the MIF receptors CXC chemokine receptor4 (CXCR4) and CD74, has been investigated. Meanwhile the effects of JNK MAPK activation on adhesive properties is assessed in response to P. gingivalis since a rapid and transient activation of the JNK pathway was triggered by P. gingivalis. In addition, the results are further confirmed in apolipoprotein E-knocked out mice inoculated with P. gingivalis. Since the vascular endothelium is essential for the adhesion of monocytes during atherogenesis, studies aimed at the impact of MIF on adhesive properties of endothelial cells pretreated by P. gingivalis may elucidate the role of this organism in atherosclerosis. The knowledge of MIF may help us make detailed treatment programme in clinic.

作为牙周炎重要致病菌，牙龈卟啉单胞菌(Pg)是促进动脉粥样硬化(AS)发生的重要因素之一。巨噬细胞移动抑制因子(MIF)可促进细胞间黏附分子(ICAM)-1表达、使血管内皮细胞(VEC) 黏附增加，在AS发生发展中具有重要作用。前期工作发现Pg感染VEC使ICAM-1表达增高、与单核细胞黏附增强，并可活化JNK通路。但迄今为止，关于MIF在Pg增强VEC黏附活性中的作用、Pg是否通过MIF调控ICAM-1表达以及通过哪个途径活化JNK尚不清楚。本项目以MIF为切入点，从细胞和动物实验两个角度，利用siRNA基因沉默、MIF表达载体转染技术以及小鼠AS模型，研究MIF在Pg增强VEC黏附活性中的作用；明确MIF是否通过与CXCR4或CD74受体相结合而参与JNK通路的调控过程。研究结果将为伴Pg感染的AS样疾病的干预治疗提供理论依据，MIF有望成为临床诊断、治疗的新靶点。

大量临床研究、流行病学观察和基础研究表明，慢性牙周感染对动脉粥样硬化样疾病的发生发展起促进作用。但是牙周感染通过哪些途径、以何种方式、以及促进动脉粥样硬化样疾病发生发展的具体机制尚不明了。我们首先采用体外研究探讨牙周致病菌——牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）——感染诱导血管内皮细胞发生炎症反应的过程，分析在动脉粥样硬化早期病变中调控细胞黏附活性增强的关键炎症因子及其发挥的作用。.我们验证了P. gingivalis 感染血管内皮细胞后，①炎症因子 MIF浓度显著升高；②诱导血管内皮细胞ICAM-1表达增强，并通过ICAM-1介导的途径增加单核细胞—血管内皮细胞黏附，炎症因子巨噬细胞移动抑制因子（macrophage migration inhibitory factor, MIF） 的特异拮抗剂ISO-1明显降低细胞间黏附分子(intercellular adhesion molecule, ICAM)-1 表达，过量的人重组MIF则可使降低到ICAM-1的表达得到回复；③荧光显微镜下观察单核细胞与其黏附的水平显著升高；MIF的特异性拮抗剂ISO-1可抑制MIF，使单核细胞——血管内皮细胞黏附水平下降，外源性人重组MIF能够中和ISO-1的作用，回复单核细胞——血管内皮细胞黏附水平。 ④对细胞表面的CD74或CXCR4受体进行检测分析，发现特异性阻断MIF的受体CD74或CXCR4后，ICAM-1蛋白及mRNA表达均减少，单核细胞——血管内皮细胞黏附水平降低。P. gingivalis 感染细胞伴随着JNK通路活化。 ⑤ 体内研究验证体外研究的结果，发现： MIF作为一种炎症因子参与慢性牙周炎的发展进程，其表达水平可作为炎症指标辅助判断慢性牙周炎活动性及炎症程度。 慢性牙周炎组血清中MIF及单核细胞趋化蛋白-1浓度较牙周健康者明显升高；在牙龈上皮MIF呈阳性表达，且在慢性牙周炎组表达水平最高，牙周基础治疗后，MIF表达水平显著下降。.总之体内、体外研究结果显示：在P. gingivalis增强血管内皮细胞黏附活性的过程中，MIF调控细胞间黏附分子1表达，进而影响单核细胞-血管内皮细胞黏附效率。我们的研究为阐明P. gingivalis感染促进AS样疾病发生提供理论依据。