TSP-1-CD47在ROS介导百草枯致肺纤维化中的作用

The Role of TSP-1-CD47 in ROS-mediated Pulmonary Fibrosis Induced by Paraquat

目的以人源A549细胞为研究对象，体外构建百草枯（Paraquat，PQ）细胞模型，探讨血小板反应蛋白（Thrombospondin，TSP）及其受体CD47在PQ致肺纤维化中的作用。方法体外培养人源A549细胞，实验分为正常组、PQ组、Anti—TSP1组（PQ＋终浓度为10μg／ml的TSP1中和抗体）。采用CCK8法检测不同浓度PQ（50、100、200、400、600、800、1000μmol/L）在不同时间（12、24、48h）对细胞存活率的影响，以细胞半数存活率为标准筛选出作用于后续试验的浓度和时间点；倒置显微镜观察细胞形态变化；采用酶联免疫吸附（Enzyme Linked Immunosorbent Assay，ELISA）法测定纤维连接蛋白（Fibronectin，FN）、I型胶原的表达量；免疫细胞化学（Immunocytochemistry，ICC）法观察TSP-1、CD47的蛋白表达情况；免疫荧光双染（Immunonuorescence double staining）察TSP-1、CD47共表达；实时荧光定量PCR（Real Time PCR，RT-PCR）检测TSP-1、CD47mRNA的表达量，Western印迹法（Western Blot，WB）检测TSP-1、CD47蛋白表达；流式细胞仪检测细胞内活性氧（Reactive Oxygen Species，ROS）水平。结果PQ作用时间一定时，细胞活性随PQ浓度增加而降低。正常组细胞间连接紧密，呈鹅卵石状，排列整齐，岛状或成团生长；PQ组细胞随着PQ浓度增加，细胞间隙逐渐增大，形态逐渐变得不均一，伸出不规则突起，呈纺锤体形或长梭形。随着PQ作用浓度增加，与正常组比较，PQ组FN、Ⅰ型胶原的相对表达量逐渐增多，差异有统计学意义（P〈0．05），以200μmol／L时最为明显，差异有统计学意义（P〈0．05）。随着PQ作用浓度增加，与正常组比较，PQ组TSP-1、CD47的mRNA水平表达上调、蛋白表达量明显增加，差异有统计学意义（P〈0．05），以200μmol／L时最为明显。免疫荧光显示TSP-1、CD47在细胞质中存在共表达，差异有统计学意义（P〈0．05）。与正常组比较，PQ组细胞ROS水平在24h时明显升高，差异有统计学意义（P〈0．05）。与PQ组比较，Anti—TSP1组细胞活性明显增加，差异有统计学意义（P〈0．05），形态趋向正常细胞形态变化；TSP-1、CD47的RNA水平及蛋白表达量均有下降，差异有统计学意义（P〈0．05）；ROS的过表达受到抑制，差异有统计学意义（P〈0．05）；FN、Ⅰ型胶原的相对表达量减少，差异有统计学意义（P〈0．05）。结论PQ刺激A549细胞可诱导细胞发生形态学改变，使TSP-1、CD47表达增加、FN、Ⅰ型胶原的表达量升高、ROS产生增多。TSP-1中和抗体对抗TSP-1后，可部分改善以上变化。TSP-1-CD47可能与氧化应激介导的PQ致肺纤维化过程相关联。

Objective Taking human A549 cells as the research object, to construct the paraquatinduced pulmonary fibrosis model in vitro, and to explore the,role of TSP-1 （Thrombospondin-1, TSP-1 ） and its receptor CD47 in PQ-induced pulmonary fibrosis. Methods Human A549 cells were cultured in vitro, divided into normal control group, PQ group, Anti-TSP1 group （PQ with neutralizing anti-TSP1 antibody at a final concentration of 10 μg/ml）. A549 cells were stimulated with different concentrations （50, 100, 200, 400, 600,800, 1 000 μmol/L） for different time（ 12, 24, 48 h）, and then CCK8 method was used to detect the cell viability to screen out the concentration and time of half cell viability. The subsequent test will be performed at this concentration point.The morphology of the cells was observed under inverted microscope. The expression levels of Fibronectin （FN） and type I collagen were determined by Enzyme Linked Immunosorbent Assay （ELISA）. Immunocytochemistry （ICC） and Immunofluorescence （IF） were used to observe the expression of TSP-1 and CD47 protein and the co-expression.The mRNA expression of TSP-1 and CD47 was detected by Real Time PCR （RT-PCR）. The protien expression of TSP-1 and CD47 was detected by Western Blot （WB）. The levels of Reactive Oxygen Species （ROS） were measured by flow eytometry. Results Before neutralizing anti- TSP1 antibody intervention： （1） When the time of PQ was constant, the cell viability decreased with the increase of PQ concentration. （2） The cells in the control group were closely connected, cobble-like, arranged neatly; with the increase of PQ concentration, the cell gap of PQ group gradually increased, spindle shape or long spindle shape. （3） With the increase of PQ concentration, the relative expression of FN and I collagen in PQ group was gradually increased compared with the control group in a concentration-dependent manner, and 200 μmol/L is the most obvious. （4）Compared with the control group, the mRNA level and the protein expression of TSP-1 and CD47 in PQ group was significantly increased, and 200 μmol/L is the most obvious, and Immunofluorescence showed they were co-expression in cytoplasm. （5）Compared with the normal group, the level of ROS in A549 cells was significantly increased at 24 h after PQ stimulation. （6） Compared with PQ group, the cell viability of Anti-TSPI group was significantly increased, and the morphology changed to normal cell morphology, and the mRNA level and the protein expression of TSP-1 and CD47 decreased,and the overexpression of ROS was inhibited, and the relative expression of FN and I collagen decreased. Conclusion PQ stimulation induced morphological changes of A549 ceils, increased expression of TSP-1, CD47, FN and type I collagen, and increased production of ROS.Neutralizing anti-TSP1 antibodies against TSP-1 can partially improve the above lesions. TSP-1-CD47 may be associated with oxidative stress-mediated PQ-induced pulmonary fibrosis.