| Object:Taking human A549 cells as the research object,to construct the paraquat-induced pulmonary fibrosis model in vitro,and to explore the role of TSP-1 and its receptor CD47 in PQ-induced pulmonary fibrosis and its possible mechanism.Method: Human A549 cells were cultured in vitro,divided into normal control group,PQ group,blank group for the first.A549 cells were stimulated with different concentrations(50u M,100 μM,200 μM,400 μM,600 μM,800 μM,1000 μM)for different time(12 h,24 h,48 h),and then CCK-8method 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.After that,divided into control group and PQ group.The PQ group consisted of three concentrations(50 μM,100 μM,200 μM)for 24 h.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-1and 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 cytometry.Finally,divided into control group,PQ group,Anti-TSP1 group(PQ + neutralizing anti-TSP1antibody).The PQ group consisted of one concentrations(200 μM)for 24 h,and the Anti-TSP1 group was co-cultured with pre-added 10 μg/mlneutralizing anti-TSP1 antibody and 200 μM PQ.The cell viability,morphological changes,TSP1,CD47 mRNA and protein level,intracellular ROS levels,extracellular matrix FN and type I collagen expression were observed again.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,and it was time-dependent.Compared with the control group,the viability of cells after 50μM PQ was not statistically significant(P> 0.05),the rest were statistically significant(P <0.05).(2)The cells in the control group were closely connected,homogeneous in shape,cobble-like,arranged neatly,island-like or group-like growth,adherent strong,strong ability to proliferate;with the increase of PQ concentration,the cell gap of PQ group gradually increased,the morphology gradually became heterogeneous,irregular,protruding irregular protrusions,spindle shape or long spindle shape,proliferation ability gradually weakened.(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 μ M is the most obvious.(4)Compared with the control group,the mRNA levelof TSP-1 and CD47 in PQ group was significantly increased,and 200 μ M is the most obvious.(5)Compared with the control group,the protein expression of TSP-1 and CD47 in PQ group was significantly increased,which was the most obvious at 200μM,and they wereco-expression in cytoplasm.(6)Compared with PQ group,the cell viability of Anti-TSP1 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 ofROS was inhibited,and the relative expression of FN and I collagen decreased after anti-TSP1 antibody intervention,and all were statistically significant(P<0.05).Conclusions:(1)200 μM PQ stimulated A549 cells for 24 hours to construct pulmonary fibrosis model successfully in vitro.(2)PQ stimulates A549 cells to induce TSP-1 and CD47 expression,and co-expression in cytoplasm.(3)PQ induced oxidative stress in A549 cells.(4)Anti-TSP1 antibody reduced the TSP-1,CD47 mRNA and protein expression,inhibited oxidative stress,decreased FN,type I collagen expression.(5)TSP-1-CD47 may be involved in PQ-induced pulmonary fibrosis by inducing oxidative stress. |
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