| Background: According to the International Standard Organization(ISO)definition: air pollution generally refers to the human activity or natural process caused by certain substances into the atmosphere,when a certain amount of time,after a certain period of time to cause health hazards of human and other species,reduction of comfort and other discomfort and other phenomena.In air polluted particles(particulate matter),the aerodynamic equivalent diameter is less than 2.5 microns.The particles that can enter the human respiratory system are called PM2.5.It can be suspended in the air for a long time.The higher the concentration in the air,the more serious the air pollution is.Although PM2.5 is only a few components in the earth's atmosphere,it has an important impact on air quality and visibility.Compared with the coarser atmospheric particles,PM2.5 has small particle size,large area and strong activity.It is easy to accompany toxic and harmful substances(such as heavy metals,microorganisms,etc.),and has a long stay in the atmosphere and long transport distance,so it has a greater impact on human health and the quality of the atmosphere.In air polluted particles,PM2.5 has the greatest harm to human health.It can escape the filtration and cleanliness of the mucous membrane defense system on the upper respiratory tract and go deep into the alveoli.Macrophages in the lung play a key role in the defense of PM2.5.Macrophages endocytosis the PM2.5 package through its surface protrusion,pseudo foot or some pattern recognition receptors,prompting the release of some cytokines and chemokines,causing pulmonary inflammatory response and oxygenation stress response.These reactions are necessary for the lung to resist invasive substances,but excessive PM2.5 stimulation will lead to the imbalance of inflammatory response and oxidative stress,which will lead to respiratory diseases.Tuberculosis is a chronic infectious disease of the lungs by Mycobacterium tuberculosis(Mtb).Mtb can invade all organs of the whole body,and the harm to human health is very serious.Macrophage mediated immune response is an important defense system for anti-Mtb infection.However,there are few studies on the effect of PM2.5 on macrophage phagocytosis of Mtb.This paper aims to explore the effect of PM2.5 on macrophage immune function and the ability of phagocytosis of Mtb.Objective: To investigate the effect of PM2.5 on the function of macrophage and the ability of phagocytic Mycobacterium tuberculosis Methods:(1)Induction of macrophage The THP1 cells with better status were transferred to the cell density of 5 * 105/ mL with a complete 1640 culture medium containing 10% fetal bovine serum.After 24 hours of induction with PMA(80ng/ml),the cells will grow on the wall,and the morphology becomes irregular.This is the macrophage.(2)Cultivation of Mycobacterium tuberculosis strain H37 Ra H37Ra(H37Ra-Red),which is used to express red fluorescent protein(GFP),is constructed and provided by our laboratory teachers in this experiment.H37Ra-Red is a high requirement for aerobic bacteria,and the growth is slow.In this experiment,the culture of Sutong culture with 10% calf serum was cultivated at 37?.After about 15 days,the red film membrane was produced in the liquid surface.The red was red fluorescent protein expressed in H37Ra-Red.After about 30 days,the membrane became thickened and wrinkled,and the green bean shaped membrane was inoculated in PBS for inoculation.On the 7H11 solid slope culture medium,the inoculation ring was snakes on the slope,and it was cultivated at 37? for about 30 days,and the colonies were like cauliflower like growth.(3)Preparation of PM2.5 suspension A PBS suspension with a concentration of 2mg/ml and 4mg/ml from the Beijing region of China and the Baltimore region of the United States of America was placed at 4 ? for storage and used in a week.(4)Detection of TLR4 After stimulating macrophages for 12 hours and 24 hours by 0,50,and 200ug/ml PM2.5 in two regions,the expression of TLR4 was detected by flow cytometry.(5)Detection of ROS After stimulating macrophages for 12 hours and 24 hours by 0,50,and 200ug/ml PM2.5 in two regions,the production of ROS was detected by fluorescence microscopy and flow cytometry respectively.(6)Detection of apoptosis rate After stimulating macrophages for 12 hours and 24 hours by 0,50,and 200ug/ml PM2.5 in two regions,the cells were stained with PI/Annexin V-FITC and the cell apoptosis was detected by flow cytometry.(7)Detection of phagocytosis by macrophages phagocytic H37Ra-Red After the macrophages were stimulated by 0,50,and 200ug/ml PM2.5 in two regions for 12 hours and 24 hours,the macrophages were added to H37Ra-Red at 37?for 12 hours,and the fluorescence microscope was used to observe the phagocytosis of H37Ra-Red by macrophages.(8)Detection of Nrf2,TLR4,IL12P35 and IL12P40 After stimulating macrophages by 0,50,and 200ug/ml PM2.5 in two regions for 12 and 24 hours,the expression of TLR4,IL12P35,IL12P40 and Nrf2 genes was detected by fluorescence quantitative PCR,and the expression of Nrf2 protein level was detected by Western-blot technology.Results:(1)After different concentrations of PM2.5 interfering with macrophages for 12 hours,the production of ROS increased in a dose-dependent manner with the increase of PM2.5 concentration.However,after 24 hours,the production of ROS increased at first and then decreased with the increase of PM2.5 concentration.(2)After 12 and 24 hours of PM2.5 interfering with different concentrations of macrophages in two regions,the production of TLR4 showed a tendency to increase first and then decrease with the increase of PM2.5 concentration.(3)After 12 and 24 hours of PM2.5 interfering macrophages with different concentrations in two regions,the total cell apoptosis rate increased with the increase of PM2.5 concentration.(4)After 12 and 24 hours of PM2.5 stimulated by different concentrations of PM2.5,the ability of cells to phagocyt H37Ra-Red increased and then decreased with the increase of PM2.5 concentration.After 24 hours,the phagocytic ability of macrophages in the high concentration group of two regions was lower than that of the negative control group(P<0.05).(5)Fluorescence quantitative PCR results: two regions with different concentrations of PM2.5 interference in macrophages after 12 hours,the expression of IL12 was increased with PM2.5 concentration in a dose-dependent manner and increased expression of Nrf2 in Beijing with PM2.5 concentration increased in a dose-dependent manner increased expression of Nrf2 in Baltimore compared with negative control group expression decreased,TLR4 expression was first increased and then decreased with the increasing of PM2.5 concentration,after 24 hours,the expression of Nrf2 and IL12 with PM2.5 concentration increased firstly and then decreased,the expression of TLR4 expression decreased compared with negative control group.(6)Western-blot showed up-regulated the expression of Nrf2 in Beijing area with different concentrations of PM2.5 interference macrophages after 12 hours was dose dependently elevated after 24 hours and did not detect the expression of Nrf2,PM2.5 in Baltimore area 12,24 hours after the interference of macrophages was detected the expression of Nrf2.Conclusion:(1)Low concentration of PM2.5 can stimulate macrophage immune function and promote macrophages to phagocytosis of Mycobacterium tuberculosis.(2)High concentration of PM2.5 decreases the ability of macrophages to swallow Mycobacterium tuberculosis by damaging cell function and inducing apoptosis. |
PDF Full Text Request