牙龈卟啉单胞菌感染对单核细胞源性泡沫细胞形成的影响及其机理

Recent a growing body of evidences suggested that periodontitis is a independent risk factor for cardiovascular disease(CVD).To discover the biological mechanism between periodontal disease and CVD has been a new research area.In recent years seroepidemiology、animal and molecular biology studies have suggested that P.g,which is a major etiologic agent in the pathogenesis of periodontal disease in humans, can diffuse into bloodstream and invade into the arterial wall and human coronary endothelial cells, then upregulate the expression of cell adhension molecules in endothelial cells. These evidences suggested that P.g infection may be associated with the development of atherosclerosis. In light of the formation of macrophage foam cells in the arterial intima is a hallmark of early lesion formation in atherosclerosis and the imbalance of cholesterol metabolism home ostasis in macrophage contributes to foam cell formation,we have provided evidence to confirm that infection by P.g can induce foam cell formation by human monocyte-derived macrophages.And P.g may play a important role in formation of macrophage foam cells in cardiovascular tissue. However, detailed mechanisms have not fully been studied. In order to investigate potential role of P.g in the biological mechanism between periodontitis and CVD , the model of infection of monocyte-derived macrophages by P.g will be established in vitro in the present study:1)To observe the effect of P.g infection on the formation of THP-1-derived foam cells in vitro. Lipid droplets in cytoplasm were observed by oil red O staining.The Contents of intraeellula cholesterylesters were detected by enzymic chromatometry;2) To investigate the roles of the expressions of mRNA and protein of key genes involved in macrophage cholesterol metabolism in P.g-induced foam cell formation by PCR and Western blot;3) To discuss the pathways of P.g-induced foam cell formation.These research may provide new insights for the development and progression of atheroselerosis initiated by P. g infection and supply evidences for the intervention and treatment of CVD in the early stage.

最新资料支持牙周炎是心血管疾病（CVD）的独立危险因素，两者相关联的生物学机制备受关注。近来血清流行病学、动物和细胞分子学研究发现:作为慢性牙周炎主要致病菌之一的牙龈卟啉单胞菌（P.g）可入血并侵入血管壁，参与动脉粥样硬化（AS）的发生发展。鉴于巨噬细胞形成泡沫细胞在AS发生发展中的关键性作用，申请人通过前期的探索性研究工作初步证实:P.g感染单核细胞源性巨噬细胞后可显著促进其形成泡沫细胞。但其机理尚不清楚，亦未见相关报道。本研究拟在前期已建立的P.g感染人单核细胞源性巨噬细胞体外模型的基础上，采用脂质染色和胆固醇测定法观察P.g感染单核细胞源性巨噬细胞后对泡沫细胞形成的影响；从基因和蛋白水平了解P.g感染后巨噬细胞胆固醇代谢关键分子的表达变化；探讨PPARs信号通路在P.g促进泡沫细胞形成中的作用，旨在阐明P.g感染在牙周炎与CVD相关性中的生物学机制，为CVD的早期干预提供实验依据。

一、背景：牙周炎是心血管疾病（CVD）的独立危险因素之一，但是两者相关联的生物学机制还不甚清楚。本项目围绕P.g-LPS对泡沫细胞形成的影响，研究胆固醇代谢关键分子的表达变化；探讨PPARs信号通路在其中的作用，部分阐明了P.g感染在牙周炎与CVD 相关性中的生物学机制。二、方法： 1、诱导THP-1单核细胞分化为巨噬细胞。在负荷50 μg/ml LDL的条件下，分别用不同浓度的P.g-LPS与巨噬细胞共孵育48 h；1 μg/ml P.g-LPS与巨噬细胞共孵育不同时间。观察和检测胞浆内脂滴和细胞内胆固醇酯含量的变化；检测胆固醇代谢关键分子基因和蛋白表达。2、探讨P.g-LPS促进单核细胞源性巨噬细胞形成泡沫细胞的信号传导通路。THP-1源性巨噬细胞在负荷LDL的条件下，随机分为以下6组：空白对照组、P.g-LPS感染组、P.g-LPS感染＋PPARα 激动剂组、PPARα 激动剂组、P.g-LPS＋PPARγ 激动剂组、PPARγ激动剂组。从形态学和生化学的角度观察PRARa激动剂对P.g-LPS诱导的THP-1源性泡沫细胞形成的影响。三、结果：1. 160 nmol/L PMA作用48h后可诱导THP-1细胞成为巨噬细胞。2. P.g-LPS组胞内脂滴显著增多，CE/TC比值明显增加。随着P.g-LPS浓度升高，细胞内的CE/TC的比值也逐渐升高，当P.g-LPS浓度为10μg/ml时达到峰值。随着1 μg/ml P.g-LPS孵育时间的延长，胞内脂滴逐渐增多、CE/TC比值逐渐升高，当P.g-LPS孵育时间72h时，细胞内红色脂滴最多。3.随着P.g-LPS浓度的增加，CD36 和ABCG1的mRNA和蛋白表达均逐渐下降；胞内ACAT1的mRNA和蛋白表达均逐渐升高。细胞内LOX-1的mRNA和蛋白表达无明显变化。4.随着1μg/ml P.g-LPS下降CD36 和ABCG1的mRNA和蛋白表达，72h组最低。ACAT1的mRNA和蛋白表达升高，72h组最高 ；细胞内LOX-1的mRNA和蛋白表达无明显变化。5.在给予不同浓度的PPARα和PPARγ激动剂干预后，泡沫细胞明显减少。四、结论：P.g-LPS通过上调ACAT1及下调CD36和ABCG1，导致巨噬细胞内胆固醇代谢紊乱，促进细胞沫细胞的形成。PPAR通路可能参与其中。