牙龈炎症环境中环孢素A阻断亲环素A与CD147结合途径探讨

Although complex interactions among mediators of tissue remodeling and inflammation may be involved in CsA induced gingival overgrowth (CIGO), the exact mechanism is not fully understood. Cyclophilin A (CyPA) is best known as ubiquitously distributed intracellular protein which was first recognized as the host cell receptor for CsA. However, during the course of inflammatory responses, CyPA is released into extracellular tissue spaces by both live and dying cells. The secreted CyPA has multiple functions in chemotaxis and cell signaling cascade through its cell surface receptor, CD147. CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a regulator of matrix metalloproteinase (MMP) production and activation. MMPs degrade most, if not all, components of the extracellular matrix (ECM) and basal membranes. The interactions of extracellular CyPA and CD147 have been well documented in some studies. These studies show an important contribution of CyPA-CD147 interactions to the initiation and/or progression of inflammatory responses via recruitment of leucocytes into inflamed tissues and stimulation of MMP production. All these roles of CyPA-CD147 interactions can be blocked by CsA. Considering the contribution of CyPA to both inflammatory responses and MMPs production in the extracellular environment, we hypothesize that CsA can block CyPA release from cells, decrease expression of CD147 and interrupt extracellular CyPA-CD147 binding in the inflammatory environment resulting in GO. To test this we plan to evaluate the levels of gingival crevicular fluid CyPA in vivo and the expressions, levels, activities of CD147, CyPA, MMPs in vitro. Using an enzyme-linked immunosorbent assay (ELISA) approach, we propose to detect the levels of gingival crevicular fluid CyPA among CIGO patients, periodontitis patients and normal controls. Our in vitro study aim to evaluate the effect of CsA on the levels of extracellular CyPA, cell surface expression of CD147, expression and activity of MT-1 MMP, TIMP-2 and MMP-2 from both independent and co-culture of human gingival fibroblasts and macrophages in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). As control, CyPA, CD147 and MMPs are also evaluated in both independent cultures and co-cultures without CsA and Pg-LPS. The levels of CyPA in the supernatants of independent cultures and co-cultures are measured by ELISA. Cell surface expressions of CD147 are detected by flow cytometer. The expressions of MMPs mRNA are measured by qRT-PCR. Zymography is selected to evaluate the activities of MMP-2 in the supernatants of independent cultures and co-cultures. In addition, we propose to evaluate the effects of rhCyPA on the up-regulation of MMP-2 activities in the supernatants of independent and co-cultures. Our study may be significant in determining the ways of CyPA and CD147 binding blocked by CsA in the inflammatory environment resulting in GO.

环孢素A(CsA)可以通过三条途径阻断亲环素A(CyPA)与CD147结合，导致细胞外基质过度积聚，但具体阻断途径尚不清楚。我们拟用ELISA检测健康成人、CsA诱导的牙龈肥大患者和牙周炎患者龈沟液CyPA浓度，明确CsA能否阻止细胞在炎症条件下释放CyPA；建立人牙龈成纤维细胞、巨噬细胞单独和共培养模型，经不同浓度牙龈卟啉单胞菌脂多糖和/或CsA处理，用ELISA、流式细胞术、qRT-PCR、明胶酶谱法检测上清液CyPA浓度、细胞膜CD147表达、MMP-2、MT1-MMP、TIMP-2 mRNA表达、MMP-2活性，明确CsA能否在模拟炎症环境中阻滞CyPA释放、减少细胞膜CD147表达；在培养液中先后加入不同浓度rhCyPA和CsA，通过观察MMP-2活性变化，说明CsA能否在细胞外阻断CyPA与 CD147结合。明确CsA在炎症环境中阻断CyPA与CD147结合的途径。

环孢素A诱导的牙龈增生（Cyclosporin A Induced Gingival Overgrowth, CIGO）是器官移植术后患者常见的并发症，增生牙龈不仅影响患者生存质量，龈袋内的细菌更增加免疫抑制患者系统性感染的风险。CIGO发病机制尚不明确，现有研究表明，CIGO主要表现为胶原降解减少，并且与细胞外基质中基质金属蛋白酶（Matrix Metalloproteinases, MMPs）介导的胶原代谢失衡密切相关；亲环素A（Cyclophilin A, CyPA）和基质金属蛋白酶诱导剂（Extracellular matrix metalloproteinase inducer, EMMPRIN又称CD147）的相互作用调控MMPs分泌和活化。本研究旨在明确CsA对CyPA-CD147结合途径的影响及其对下游MPPs调控的作用，通过本研究将有助于深化对环孢素A诱导牙龈增生发病机制的认识，具有重要的临床和科学意义。.本课题的研究成果主要有以下2个方面：.1、动物模型的构建：通过构建大鼠环孢素A诱导大鼠牙龈增生动物模型，应用免疫组化和Western blot检测，研究CyPA、CD147、MMP-2和MMP-9在CIGO动物模型中的表达。结果显示：CsA通过下调CyPA在牙龈组织中的表达，影响了CyPA与CD147的相互作用，下调MMP-2和MMP-9表达，减少胶原降解。.2、体外实验验证：在动物实验的基础上，建立人牙龈成纤维细胞（Human Gingival Fibroblast, HGF）、巨噬细胞（Macrophage cell，MACs）单独和共培养模型，经过牙龈卟啉单胞菌脂多糖（Porphyromonas gingivalislipopolysaccharide, Pg-LPS）和/或CsA处理后，通过检测细胞上清液CyPA浓度、细胞膜表面CD147表达情况变化等。上述细胞学实验结果进一步证实：CsA能够明显降低细胞外CyPA的浓度；而单独给予炎症因子并不会明显改变细胞外CyPA浓度。在模拟牙龈炎症环境下（即MACs和HGF共培养并同时给予LPS和CsA刺激后），细胞外CyPA表达水平较单独培养时高，说明细菌毒素、炎症细胞有促进细胞向外分泌CyPA的作用。.本课题初步揭示了CsA通过干扰CyPA-CD147的结合，降低细胞外基质中MPPs的活性，