TSP-1-CD47在ROS介导百草枯致肺纤维化中的作用

发布时间：2018-02-01 09:35

本文关键词： 百草枯纤维化血小板反应蛋白-1 CD47 氧化应激　出处：《川北医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:以人源性A549细胞为研究对象,从体外构建百草枯(Paraquat,PQ)致肺纤维化模型,初步探讨TSP-1及其受体CD47在PQ致肺纤维化中的作用及其可能的发病机制。方法:体外培养人A549细胞,首先分为正常组对照组、PQ组、空白对照组,采用CCK-8法检测不同浓度(50μM、100μM、200μM、400μM、600μM、800μM、1000μM)的PQ刺激不同时间(12h、24h、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。此后分为正常组、PQ组,PQ组下设1个时间点(24h)和3个浓度(50μM、100μM、200μM)。倒置显微镜下观察细胞形态变化;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型胶原的相对表达量;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(Immunofluorescence double staining)观察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。最后,再分为正常组、PQ组、Anti-TSP1组(PQ+TSP1中和抗体),PQ组下设1个时间点(24h)和1个浓度(200μM);Anti-TSP1组(24h)加入TSP1中10μg/ml与200μM的PQ共培养。再次观察细胞存活率、细胞形态、TSP-1和CD47基因和蛋白水平、细胞内ROS水平和FN、I型胶原表达变化。结果:1.PQ作用时间一定时,细胞活力随PQ浓度增加而降低,呈时间浓度依赖性;与正常组相比,除50μM的PQ作用12h时的细胞的活力下降没有统计学意义(P0.05)外,其余均有统计学差异(P0.05);2.正常组细胞连接紧密,形态均一,呈鹅卵石状,排列整齐,岛状或成团生长,贴壁强、增殖能力强;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、无规则,伸出不规则突起,呈纺锤体形或长梭形,排列紊乱,增殖能力逐渐减弱;3.随着PQ作用浓度增加,与正常组相比,PQ组FN、I型胶原的相对表达量逐渐增多,呈浓度依赖性,以200μM时最为明显;4.随着PQ作用浓度增加,与正常组相比,PQ刺激组TSP-1、CD47 mRNA水平表达上调、蛋白表达量明显增加,以200μM时最为明显;5.与正常组相比,PQ组TSP-1、CD47蛋白表达量明显增加,均以200μM时最为明显;TSP-1、CD47在细胞质中存在共表达;6.与正常组相比,ROS水平在24h时明显升高。加入TSP1中和抗体后,与PQ组相比,Anti-TSP1组细胞活力明显增加,形态向正常细胞形态变化;TSP-1、CD47的mRNA水平及蛋白表达量下降;ROS的过表达受到抑制;FN、I型胶原的相对表达量减少。结论:1.200μM的PQ刺激A549细胞24h能够成功构建体外肺纤维化模型;2.PQ刺激A549细胞后可诱导TSP-1和CD47的表达,且在细胞质中存在共表达;3.PQ可刺激A549细胞发生氧化应激;4.TSP-1中和抗体可下调TSP-1、CD47 mRNA水平及蛋白表达量,抑制氧化应激状态,降低FN、I型胶原表达量;5.TSP-1-CD47可能通过诱导氧化应激参与了PQ致肺纤维化过程。
[Abstract]:Objective: to establish a model of pulmonary fibrosis induced by Paraquat PQ from human A549 cells in vitro. To explore the role of TSP-1 and its receptor CD47 in PQ induced pulmonary fibrosis and its possible pathogenesis. Methods: human A549 cells were cultured in vitro and divided into normal control group and PQ group. In the blank control group, the different concentrations of 50 渭 M ~ 100 渭 m ~ (100 渭 m) ~ (100 渭 m) ~ (100 渭 m) ~ (100 渭 m) were detected by CCK-8 method. The effect of PQ (1 000 渭 M) on cell survival was observed at different time points (12h ~ 24h ~ 48h). The concentration and time point acting on the follow-up test were selected according to the standard of 50% cell survival rate. After that, they were divided into normal group (PQ group) and three concentration groups (50 渭 M), one time point (24 h) and three concentrations (50 渭 M) under PQ group. The morphologic changes of the cells were observed under inverted microscope. Enzyme Linked Immunosorbent Assay was used as enzyme linked immunosorbent assay (Elisa). The relative expression of fibronectinine fibronectin (FN) type I collagen was determined by ELISAmethod. The expression of TSP-1mCD47 protein was observed by immunocytochemistry-ICCassay. Immunofluorescence double staining was used to observe the co-expression of TSP-1 and CD47. The expression of TSP-1 and CD47 mRNA was detected by real-time fluorescence quantitative PCR(Real Time PCR-RT-PCR. Western blot was used to detect the expression of TSP-1 and CD47 protein. Flow cytometry (FCM) was used to detect the level of reactive Oxygen species in the cells. Finally, it was divided into normal group and PQ group. In the Anti-TSP1 group, the concentration of PQ TSP1 neutralizing antibody was 24 h) and the concentration of PQ was 200 渭 m. Anti-TSP1 group was co-cultured with 10 渭 g / ml of TSP1 and 200 渭 M PQ for 24 h. Cell survival rate and cell morphology were observed again. The changes of TSP-1 and CD47 gene and protein levels, ROS level and type I collagen expression in the cells were observed. Results the cell viability decreased with the increase of PQ concentration at the same time. In a time-concentration dependent manner; Compared with the normal group, the activity of the cells treated with 50 渭 M PQ for 12 h was not significantly decreased (P0.05), but there was a significant difference in the other groups. 2. In the normal group, the cells were closely connected, uniform in shape, cobbled, arranged neatly, island or agglomerate, strong adherent and strong proliferative ability; In PQ group, with the increase of PQ concentration, the cell gap gradually increased, the morphology became uneven, irregular protruding, spindle-shaped or long fusiform, disordered arrangement, and the proliferation ability gradually weakened. 3. With the increase of PQ concentration, the relative expression of FNNI collagen in PQ group gradually increased in a concentration dependent manner, especially at 200 渭 M. 4. With the increase of PQ concentration, the expression of TSP-1 + CD47 mRNA was up-regulated and the protein expression was significantly increased in PQ stimulated group, especially at 200 渭 M. 5. The expression of TSP-1 and CD47 protein in PQ group was significantly higher than that in normal group, especially at 200 渭 M. There was coexpression of TSP-1 and CD47 in cytoplasm. 6. Compared with the control group, the level of Ros increased significantly at 24 h. After adding TSP1 neutralizing antibody, the cell viability of Anti-TSP1 group was significantly higher than that of PQ group. Morphologic changes to normal cells; The mRNA level and protein expression of TSP-1 CD47 were decreased. The overexpression of ROS was inhibited. Conclusion the lung fibrosis model of A549 cells can be successfully constructed by 1. 200 渭 M PQ stimulation for 24 h. 2. PQ could induce the expression of TSP-1 and CD47 in A549 cells, and there was coexpression in the cytoplasm of A549 cells. 3. PQ stimulated oxidative stress in A549 cells. 4. TSP-1 neutralizing antibody could down-regulate the expression of TSP-1 CD47 mRNA and protein, inhibit oxidative stress and decrease the expression of FNN-1 collagen. 5. TSP-1-CD47 may be involved in the process of pulmonary fibrosis induced by PQ by inducing oxidative stress.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R595.4;R563

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