类胰蛋白酶激活心房成纤维细胞的机制研究

It is well recognized that atrial fibros is a substrate for atrial fibrillation, but the precise mechanisms remain unclear. Our pilot study revealed that ①the tryptase level of left atrium from patinetns with AF was significantly increased and ② tryptase stimulate atrial fibrobalst proliferation. Therefore we hypothesized that tryptase activates atrial fibroblast palys a povital role in atrial fibrosis. Based on our hypothesis, in the present study, cultured rat atrial fibroblast will be incubated with tryptase, tryptase specific inhibitor. The proliferation of fibroblast will be determined using BrdU incorporation, cell migration will be assessed using Transwell Chanmber method. Masson trichrome, Sirius red and Western-blot analysis will be used to evaluated the parameters of extracellular matrix remodeling. The levels TGF-β,IL-6 and ET-1 will also be determined using real-time PCR and Western-blot method.To elucidate the underling mechnisms, the cultured atrial fibroblasts will be treated with protease activated receptor-2 antagonist, platelet-drive growth factor receptor antibody and peroxisome proliferator-activated receptor γ antagonist,the proliferation, migration and collagen syntesis will be determined. These meaningful results will enlarge our scope about the mechanism of atrial fibrosis, and may shed light on the prevention and treatment of atrial fibrillation.

心房纤维化是房颤发生与维持的重要基质之一，但其确切机制尚不清楚。本课题组的前期研究结果发现①房颤患者心房组织类胰蛋白酶水平升高；②类胰蛋白酶刺激心房成纤维细胞增殖。因此我们假设类胰蛋白酶激活心房成纤维细胞可能是心房纤维化形成的重要机制之一。基于上述假设，本研究拟体外培养心房成纤维细胞，给予类胰蛋白酶、类胰蛋白酶特异性抑制剂（APC2095），采用BrdU法评价成纤维细胞增殖，Transwell小室法评价细胞迁移，免疫组化及Westren-blot检测细胞外基质重构，实时定量PCR及Western-blot检测TGF-β、IL-6、内皮素-1等水平；并予以细胞蛋白酶活化受体2特异性阻滞剂（FSLLRY）、血小板源性生长因子受体抗体、过氧化增殖体激活受体γ阻滞剂干预，明确类胰蛋白酶激活心房成纤维细胞的机制。这些结果将促进对心房纤维化机制的认识，进而为更好的预防、治疗房颤提供重要的理论依据。

本研究在为期三年的研究期内，选择9周龄雄性Sprague-Dawley大鼠，全麻无菌条件下获取大鼠心房组织，分离培养大鼠心房成纤维细胞，体外原代培养。选择生长良好的2-4代心房成纤维细胞，给予类胰蛋白酶、APC366、FSLLRY、APA5及BADGE干预24、72小时后，收集细胞。采用Edu检测法评价对心房成纤维细胞增殖的影响、Transwell小室法检测心房成纤维细胞的迁移能力变化；采用Masson染色及免疫细胞化学及Western-blot检测对心房成纤维细胞纤维化指标的影响。. 本研究结果发现：（1）类胰蛋白酶增强心房成纤维细胞的增殖、迁移能力，加重细胞纤维化。（2）类胰蛋白酶通过激活蛋白酶活化受体2（PAR2）促进成纤维细胞增殖，加重细胞纤维化。（3）类胰蛋白酶通过激活血小板源性生长因子受体促进心房成纤维细胞迁移。. 结论：类胰蛋白酶激活心房成纤维细胞，促进心房成纤维细胞增殖、迁移，加重心房纤维化；其作用机制为激活蛋白酶活化受体2及血小板源性生长因子受体。