The Role Of Integrins And FNs Proteins In The Migration Of Macrophages

The harm of smoking to human health is well known.According to statistics,the number of deaths due to smoking can reach 2.5 million every year.Smoking has become a major human health issue in the word.Related studies have shown that smoking can lead to the blockage of macrophages in the body,reduce the ability of phagocytosis and migration of macrophages,and reduce the ability of body to resist infection.Macrophages are immune cells of human body and participate in the pathogenesis of many diseases of human body.In recent years,macrophages are also regarded as the main therapeutic targets,and the disease can be improved by regulating their functions.In this study,two cell models,primary macrophage and mouse macrophage cell line RAW264,were used as research objects.The effects of nicotine on the dynamics and morphology of macrophages were detected at the living cell level by high content live cell imaging analysis system;Localization and quantification of integrins and FNs proteins by immunofluorescence staining and scanning electron microscopy;Detection of expression and binding of several integrin proteins on cell surface using broad-spectrum kit;Analysis of interaction between different integrins and FN-a protein in RAW264 cells by ELISA;Effect of nicotine on the expression of integrins and FNs proteins in macrophages by Western Blot;The mRNA expression of integrins in macrophages were detected by real-time fluorescence quantitative PCR.The results showed as follows:1.In our high content analysis,in conventional culture dishes,nicotine reduces the speed and distance of cell movement,decreases the length and area,and changes the angle of motion.In ECM dishes,the area and length of primary macrophages increased after nicotine treatment,and the distance of movement decreased.There was a certain difference in the speed of cell movement at different times.There was a significant difference in the area,length,speed and distance of RAW264 when exposed to nicotine for different time intervals.There was no significant difference in motor angle in both no matter primary macrophages or RAW264 cells in ECM culture dish.2.The results of immunofluorescence and scanning electron microscopy showed that,the expression of integrins in macrophage is mostly distributed in the cells of pseudopodium,and a partial match with the extracellular matrix protein FN-?.The distribution of FN-a protein in the migration path of cells and pseudopodia.The expression of integrin ?1,integrin av and FN-a proteins were decreased after nicotine treatment.3.Detection of integrins and FNs expression patterns,before primary mouse macrophages were treated with nicotine,there was a relatively high expression of integrin ?2,integrin ?5,integrin ?1 and integrin ?2 in RAW264 cells,Furthermore,there is a certain correlation between alpha integrin and beta integrin.Integrin ?3,integrin ?5,integrin ?1 and integrin ?4.have a strong interaction with FN-a protein.4.The results of Western Blot indicated that,integrin ?1 protein was up-regulated at 36 h after exposure to nicotine,and then down-regulated gradually at 72 h;Integrin av protein decreased gradually from 0 h to 36 h with nicotine treatment,and 48 h was significantly up-regulated;There is a down-regulation of nicotine in FN-3 protein for 24 h;The expression of FN-4 protein was up-regulated at 24 h and 48 h,and down-regulated significantly at 72 h;The expression of FN-F protein was down-regulated at 24 h,and then decreased at 72 h after relatively stable expression.5.Quantitative Real-time PCR display,integrin ?1 protein was up-regulated at 24 h and 48 h,then down-regulated at 72 h with nicotine treatment(P<0.001);Integrin av protein was up-regulated at 24 h and 36 h,and the expression level was relatively stable later.Conclusion:The study used High content live cell imaging analysis system,Immunofluorescence staining,Scanning electron microscopy,Integrins expression pattern detection,ELISA,Western Blot and RT-PCR to explore the effects of nicotine on the morphology and function of macrophage and the mechanism of action of Integrins and FNs in macrophages from cell,protein and mRNA levels.The results suggest that these two proteins may be involved in the regulation of macrophage migration and have some correlation,which may provide important information on cell level for the treatment of macrophage-related diseases.