TFPI-2对巨噬细胞胆固醇流入/流出通路的作用及分子机制

The formation and accumulation of foam cells resulted in the augmentation of atherosclerotic lipid core, which played the crucial roles on the occurrence and development of atherosclerotic plaques; Furthermore, the imbalance of cellular cholesterol influx and cellular cholesterol efflux in macrophages was the key to foam cells formation. Tissue factor pathway inhibitor 2 (TFPI-2) is a member of the Kunitz-type family of serine proteinase inhibitor. Our recent researches suggested TFPI-2 had the effects anti-atherosclerosis in vivo, and further its inhibited effects on macrophage-derived foam cells formation in vitro. Based on these results, our further study will be carried out as follow: Initially, we will determine TFPI-2's roles on the foam cells formation of macrophages with adenovirus vector or siRNA's interfered technique unregulated/ downregulated the expression of TFPI-2 respectively in macrophage-derived foam cells model; Meanwhile we will observe what roles TFPI-2's upregulation or downregulation play on some important genes of cellular cholesterol influx/efflux pathway, including the scavenger receptor CD36, peroxisome proliferator-activated receptor gamma (PPARγ), the nuclear receptors liver X receptor alpha (LXRα), ATP-binding cassette transporters A1 and G1 (ABCA1, ABCG1) and Apolipoprotein A-I (apoA-I); Moreover, cellular cholesterol influx/efflux pathway will be intervened by its agonist or antagonist, and then the effects on expression of TFPI-2 and on the foam cells formation of macrophages will be observed, which is in order to reveal further the molecular mechanism of TFPI-2's regulation on balance of cellular cholesterol influx/efflux and foam cells formation. Our prospective results will confirm the fact that TFPI-2 have the anti-atherosclerotic effects, and will reveal the molecular mechanism of TFPI-2's regulation on balance of cellular cholesterol influx/efflux.

泡沫细胞形成堆积、脂质核心增大是动脉粥样硬化（AS）斑块发生、发展的决定因素，而细胞内胆固醇流入和流出平衡失调是导致泡沫细胞形成的关键；组织因子途径抑制物2（TFPI-2）为丝氨酸蛋白酶抑制物家族成员，本课题组新近发现TFPI-2在体内具有抗AS作用，进一步在体外发现其可抑制巨噬细胞泡沫化。据此，本课题首先应用腺病毒载体和siRNA干扰技术分别上调/下调TFPI-2表达，确定其对巨噬细胞泡沫化的作用；并进一步分析其对巨噬细胞胆固醇流入/流出通路中关键基因CD36、PPARγ、LXRα、ABCA1、ABCG1及apoA-I的调节作用；然后干预胆固醇流入/流出通路，观察其对TFPI-2表达的效应以及对巨噬细胞泡沫化作用，以进一步阐明TFPI-2调节巨噬细胞内胆固醇流入/流出平衡、抑制泡沫细胞形成的分子机制。研究结果将确立TFPI-2的抗AS作用，揭示其调节细胞内胆固醇流入和流出平衡的分子机制。

泡沫细胞形成堆积、脂质核心增大是动脉粥样硬化（AS）斑块发生、发展的决定因素，而细胞内胆固醇流入和流出平衡失调是导致泡沫细胞形成的关键。组织因子途径抑制物2（TFPI-2）为丝氨酸蛋白酶抑制物家族成员，课题组前期发现TFPI-2在体内具有抗AS作用，并在体外发现其可抑制巨噬细胞泡沫化。本课题证实了TFPI-2对巨噬细胞泡沫化的作用并初步探讨了其作用的分子机制。.首先，用U937、THP-1及RAW264.7细胞株分别构建上调/下调TFPI-2表达的稳转细胞株，油红”O”及Filipin染色证实上调TFPI-2表达可抑制巨噬细胞泡沫化，而下调则加剧泡沫化；HPLC定量检测泡沫细胞胆固醇含量，也确证了上述变化。其次，分析TFPI-2对胆固醇流入/流出通路中关键基因的作用发现：TFPI-2表达上调后，胆固醇流入相关基因SRA、CD36、PPARγ、RXRα表达下降，胆固醇流出相关基因SR-BI、LXRα、ABCA1、ABCG1表达上升；而下调TFPI-2水平后，上述基因出现反向变化。再次，进一步采用LXRα激动剂、PPARγ拮抗剂干预后发现，TFPI-2调节胆固醇流入/流出通路的作用是通过p38 / JNK信号通路实现的：TFPI-2上调后，NF-κB表达受抑制，p38、JNK磷酸化增加， SRA、CD36、PPARγ、RXRα表达下降，而SR-BI、LXRα、ABCA1、ABCG1表达上升；反之，TFPI-2下调后也可反证这一调节作用。.另外，课题组力图寻找TFPI-2调节巨噬细胞泡沫化直接作用的受体蛋白。采用蛋白质芯片筛选可能与其相互作用的蛋白，结果发现：八种蛋白质与TFPI-2发生相互作用，分别位于胞膜、胞浆和核内。进一步采用免疫共沉淀的方法发现，TFPI-2与其中的TNFRSF11A共同定位于胞膜。TNFRSF11A又称RANK，是NF-κB激活子，可调节NF-κB表达，可能与NF-κB调节泡沫细胞形成相关。因此，证实以下假说将是本课题组下一步工作的重点：即RANK是TFPI-2的膜受体蛋白，TFPI-2与RANK的相互结合竞争性地抑制了RANK与其配体RANKL的结合，从而使NF-κB表达下降，是TFPI-2抑制泡沫细胞形成的关键机制。.本研究确立了TFPI-2的抗AS作用，初步揭示其调节细胞内胆固醇流入和流出平衡的分子机制，为进一步深入研究相关机制指出了方向。