清道夫受体MARCO介导的线粒体凋亡在矽肺发病中的作用研究

Scavenger receptor in the membrane of alveolar macrophages is the pioneer against foreign particles, which paly a critical role in interaction of particles and internalization. MARCO-/- rats didin't develop silicosis after instilled silca trcheally indicated that MARCO takes an essential role in silicotic lung fibrosis, but the mechanism kept unclear. At the present study, the expression of scavenger receptor, MARCO, reactive oxygen species, potential of mitocondrial, mitochondrial membrane permeability transition, cytochrome C, caspase-9 and -3, apoptotic regulating proteins auch as Bax and Bcl-2 were determinded. The the relationship between silica-exposure history, progressive of lung fibrosis and indices mentioned above will be analyzed in order to explore the regulation of mitocondrial apoptosis mediated by MARCO in the development of silicosis. The changes of indices mentioned above will be observed after the AM from all subjects lung lavage will be treated with anti-MARCO antibody, N- acetylcysteine (ROS scavenger) and Cyclosporin A (mitochondrial membrane permeability inhibitor) in vitro, and the mechanism of AM apoptosis triggered by MARCO upregulation will be reasoned. Meanwhile in vivo study on intervention lung fibrosis by anti-MARCO antibody, N- acetylcysteine and Cyclosporin A also be conducted, the indices mentioned above, precollagen and collagenⅠand Ⅲ and lung pathology will be detected. The role of anti-MARCO antibody, N- acetylcysteine and Cyclosporin A in inhibiting lung fibrosis induced by silica will be analyzed, and the preventive and curing effects of anti-MARCO antibody, N- acetylcysteine and Cyclosporin A will be assessed and the target site will be ananlyzed. The present study will bring the scientific accordances and the new idea for preventing and cure silicosis on molecular level.

巨噬细胞（AM）膜上的清道夫受体是机体对外来颗粒物反应的先锋，在颗粒物的结合及细胞内转运过程中起重要作用，A类清道夫受体MARCO基因缺陷鼠染尘后不发生矽肺，表明MARCO在矽肺纤维化中可能起关键作用，但其作用机制尚不清楚。本研究通过检测矽肺患者AM MARCO及ROS的表达、线粒体势能及线粒体膜通透性的改变、信号分子CytC、Caspase-9和-3、凋亡调控蛋白Bax及Bcl-2的表达水平，分析职业史、矽肺期别与各指标之间的关系，探讨MARCO介导的线粒体凋亡在矽肺发生发展中的规律；采用MARCO阻断剂、ROS清除剂、线粒体通透性阻断剂对矽肺患者AM及染尘大鼠进行干预，观测上述指标的变化以及对细胞凋亡和肺纤维化程度的影响，分析MARCO的表达上调对矽肺纤维化的调控机制，评价三种抑制剂对矽肺纤维化的防治效果及作用靶点，为矽肺的预防和治疗提供科学依据和创新思路。

目的 探讨矽肺患者AM表面SR-A的表达规律及其与AM凋亡、肺纤维化程度的关系以及MARCO介导AM线粒体凋亡在矽肺发生发展中的作用。. 对象与方法 选取65例矽肺患者、5例矽肺观察对象，调查其一般情况、职业史、接尘史、疾病史等，收集其肺灌洗回收液，分离、纯化AM。将纯化后的AM分成九组：未处理组、酪氨酸激酶抑制剂组、活性氧清除剂组、抗-MARCO组、抗-MSR1组、环孢霉素A组（CsA）、N-乙酰半胱氨酸（NAC）组、Caspase-8抑制剂组、Caspase-9抑制剂组，培养24h后，采用western blot检测SR-A、线粒体信号通路有关信号分子的表达及细胞凋亡情况。.SPF级健康成年雄性SD大鼠176只按体重随机分为6组：生理盐水组、矽肺模型组、NAC组、CsA、Caspase-9抑制剂组、多聚鸟苷酸组。各组按施加干预的时间点分为预防组和治疗组，预防组大鼠造模当天、治疗组大鼠于造模28天后给予不同的处理，分别观察28天和56天后处死大鼠。流式细胞术检测AM膜电位和ROS阳性率；实时定量PCR检测Ⅰ型和Ⅲ型前胶原mRNA表达水平；免疫印迹法测定肺组织内MARCO、Bcl-2、Bax、CytC、Caspase-9和Caspase-3水平。观察肺组织病理变化。. 结果 矽肺患者AM凋亡程度、MSR1、MARCO、Caspase-9、CytC、Caspase-3高于观察对象，且随着矽肺期别增高凋亡程度加重或升高。酪氨酸激酶抑制剂和NAC能够下调Caspase-3的表达；抗MARCO和抗MSR1可上调MSR1、MARCO及Caspase-3的表达。CsA、NAC、Caspase-8抑制剂和Caspase-9抑制剂可下调Caspase-9、Caspase-3、CytC的表达。染矽尘大鼠AM表面MARCO、ROS水平、肺组织Ⅰ、Ⅲ型前胶原mRNA水平、Caspase-3、Caspase-9、Bax/Bcl-2、CytC的表达、线粒体PTP开放程度、AM凋亡率、肺纤维化程度均高于对照组。PolyG、NAC、CsA和Caspase-9抑制剂可下调染矽尘大鼠肺组织凋亡信号蛋白表达，减轻肺纤维化程度。.结论 SR-A介导的线粒体凋亡参与了矽肺发病的调控；早期采用SR-A及线粒体凋亡信号抑制剂进行预防性干预，其效果优于治疗性干预。