Study Through A Novel Model Of Human Bronchial Epithelial Wound Repair On The Role Of IL-13 And Its Downstream 15LO1 In Asthma

Objective: Asthma is a chronic inflammatory airway disease,which is considered to be a multifactorial disease caused by genetic and environmental factors.Human airway epithelial cells(HAEC),located at the junction of human body and surrounding environment,are the first line of defense of respiratory tract against external stimuli,such as microorganisms,allergens,etc.Because it is impossible to do any kind of injury in human bronchi during the experiment,cell culture is often used to study the damage and repair of epithelial cells.Among them,the air-liquid plane cell culture mode(ALI)simulated the situation in the human body to a large extent,and the cultured cells will be in the polarization state,the upper layer of the cells will differentiate with cilia.However,in ALI culture,the intercellular junctions were tight,the scratch test was unstable,and it was difficult to observe.Therefore,the repair process of bronchial epithelial cells cultured with ALI has not been studied in detail.Interleukin-13(IL-13)is an important type 2 helper T cell(Th2)type inflammatory cytokine.It is known that IL-13 is associated with airway epithelial remodeling.It has been suggested that although IL-13 can cause a series of inflammation and progression of asthma,it can accelerate the healing of airway epithelial cells and promote the repair of cell damage.In the counterpart,other studies have shown that it slows healing.15-position lipoxygenase-1(15LO1)is a lipoxygenase,one of the factors that affect epithelial cells by Th2 reaction.15LO1 catalyzes the oxidation of polyunsaturated fatty acids.15LO1 and its products are elevated in asthmatic lung tissue and cultured in vitro.Interleukin 4(IL-4)and IL-13 upregulate 15LO1.At present,there is no research of15LO1's effect on the repair of bronchial epithelial cells.The purpose of this experiment is to explore new scratches and develop new,simple and easy-to-operate injury equipment to improve the consistency of epithelial cell injury in ALI culture,so as to standardize the comparison between different disease conditions,different stimulation conditions and cell damage repair difference.At the same time,new observation methods were explored in order to obtain clear images of ALI cultured cells and observe the dynamic process of cell migration during wound repair.Using new injury instrument and new observation method,the bronchial epithelial cells of asthma patients were compared with normal cells,and the difference of their damage and repair ability was compared.The effects of 15LO1 on the migration and proliferation of human bronchial epithelial cells were evaluated.Methods: 1.The HAEC from normal participants(HC),from mild to moderate asthma patients(MMA)and from severe asthma patients(SA)were collected by bronchoscopy and cultured with ALI to obtain epithelial cells in vitro,which is close to the condition of human bronchus.And new injury instruments were developed.The method of cell scratch in ALI culture was improved.Compared with the traditional method of scratching with gun head,the new injury apparatus was easy to operate New method could obtain the similar rectangle,the same size,and the edge of the wound was straight line.2mm × 10 mm size cell scratch and healing images were collected.And large area of injury images was collected to avoid local injury.Error caused by small-scale collection could be avoided.After the regular image was obtained,the cell damage repair was observed continuously.the best observation time and interval were determined.To establish a new culture method of ALI under microscope,which can keep the state of ALI culture while observing under microscope.Take microscope photographs every 20 minutes during the maximum difference period of 24 hours,obtain continuous dynamic images of cell motion,observe the migration status of each cell,and analyze the X-axis and Y-axis coordinates of each cell at each time point by IMARIS analysis.X-axis and Y-axis coordinates of each cell at each time point were analyzed by IMARIS.Computing migration through anaconda Operation Environment in spyder software got total velocity,positive migration rate along X axis and percentage of positive healing cells.They were used to study the specific process of cell damage repair.2.In this experiment,we compared the HAECs of HC,MMA and SA in the ALI state by comparing the above new scratches with the whole situation of the injury repair in the ALI state.The effect of IL-13 on damage repair was observed,and then the effect of 15LO1 enzyme inhibitor BLX2477 on damage repair was observed under IL-13 stimulation.The cell migration was observed by using the new ALI culture method under the microscope,and each cell migration pathway was obtained,and the total cell migration rate was compared with the above-mentioned different disease conditions and different stimulation conditions.The rate of positive migration along the X axis and the percentage of positive healing cells were observed.To further compare the differences in migration direction between HC,MMA and SA cells,cell migration direction is divided into two parameters: the average angle of cell migration relative to the horizontal line(Theta ?)and the migration direction inversion rate(Kappa ?)to analyze the cell migration direction.Immunofluorescence staining was performed on HAEC after injury.The percentage of Ki67 and Ed U positive cells was counted to compare the difference of cell proliferation under different stimuli.Results: 1.The difference between the scratch of the traditional gun head and the newly developed injury instrument shows that the shape of the scratch area is irregular,and the distance between the edges of the two sides of the laceration is very different on each horizontal line,and there is a great difference in the distance between the edges of the two sides of the laceration in each horizontal line.A large,folded area of cells can be seen on the edge of the scratch.The shape of the modified scratch pattern is regular,which is similar to rectangle.The distance between the edges of the two sides of the scratch is not much different on each horizontal line,and the scratch is more stable.The image of cell scratch and healing in 2mm × 10 mm size range can be collected,and the error caused by local small area can be avoided with this large area of injury image acquisition.Continuous observation of injury repair images was taken.The normal HACE is close to complete healing in 24 hours or so.In this experiment,images were collected every 8 hours,and 48 hours were continuously collected,which guaranteed to observe the process of injury repair and healing under different conditions of disease and different stimuli.According to the overall observation of injury repair,cells were more active in injury repair within 24 hours.Under the new microscope,ALI culture system observed the migration status of cells during this period and analyzed the migration trajectory of individual epithelial cells.The specific parameters of cell migration were obtained as follows: total migration velocity,positive migration rate along X axis and percentage of positive healing cells.2.Compared with the damage repair of epithelial cells in different sources and different stimuli,100% healing was achieved in HC group at 24 hours,100% in MMA group at 40 hours,and close to healing in SA group at 48 hours.Compared with the whole situation of all samples in HC,MMA,SA group,after adding IL-13,the cell damage repair was significantly decreased.In HC,MMA group,after adding IL-13,the cell damage repair was significantly decreased,while in SA group,after adding IL-13,the cell damage repair was significantly decreased.There was no statistical significance in the reduction of cell damage repair.At the same time joining the IL-13,it is fine for each group Compared with IL-13+DMSO group,the cell damage and repair were significantly increased in DMSO and 15LO1 enzyme inhibitor BLX2477,IL-13+BLX groups,and there was significant difference between the two groups in general and in each group.The cell migration test showed that there was no significant difference in the total migration speed and the percentage of positive healing cells in the HC,MMA,SA group,but the X axis migration speed in the HC group was higher than that in the MMA and SA groups,and that in the MMA group was higher than that in the SA group.After adding IL-13,the total cell migration speed,the X-axis migration speed,and the percentage of positive healing cells were significantly decreased.At the same time as joining the IL-13,the total speed of the cell migration,the speed of the X-axis movement and the percentage of the positive-healing cells were significantly higher in IL-13+BLX group than that of the IL-13+DMSO.Cell proliferation showed that IL-13 increased the proliferation of cells in the early stage of injury,and the addition of 15LO1 enzyme inhibitor or the expression of 15LO1 decreased the effect of IL-13 on proliferation.Conclusion:1.The scratch shape of the newly developed injury instrument is regular,and the scratch is more stable.The image of injury repair was collected for 48 hours to ensure the observation of the whole process and healing of injury repair.Using the new ALI culture system under microscope,the total migration velocity,the positive migration rate,and the percentage of positive healing cells along the X axis were observed in the most active period 24 hours before injury repair.2.The repair ability of normal bronchial epithelial cells was higher than that of asthma patients.Il-13 decreased the rate of cell migration rather than cell proliferation,thus reducing the repair of cell injury.15LO1 inhibitor decreased the effect of IL-13 on damage repair.Improve cell damage repair by increasing cell migration rate.