Study On The Function Of CFTR And Epithelium Repairing In Human Bronchial Under The Infection By RSV

Objective:Respiratory syncytial virus infection is the most common in clinical diseases, and also is one of the most common respiratory tract pathogens in pediatri. After bronchial epithelial cells were infected by respiratory syncytial virus, the pathological change depends on some ion channel function activity and express density. Whether the respiratory syncytial virus restrain the expression and the function of CFTR in bronchial epithelial cells. We propose to establish the bronchial epithelial cell model in vitro infected by respiratory syncytial virus. And we will study expression and function of CFTR in bronchial epithelial cells which were infected by respiratory syncytial virus.Methods:1) Establish RSV infected bronchial epithelial cells model with airway hyperresponsiveness.Dilute the original RSV virus suspension (TCID50=10-3.82/0.1ml) at the ratio of1:100,000. Titrate the virus concentrations of RSV virus to persistently infect human bronchial epithelial cells. Establish the RSV persistently infected BEC model by measuring the RSV transcription expression level with RT-PCR method. 2) Measure cystic fibrosis transmembrane transduction regulatory factor expression levels in human bronchial epithelial cells after RSV infection Take the negative control group cells at the logarithmic phase of RSV persistently infection and the model group of cells in the transfer of1generation cells after persistently infected with RSV and stable transfer of culture, through the immunofluorescence method to detect CFTR protein level positioning and expression of the RSV persistent infection cell;use Western blotting method to detect protein expression and qRT-PCR method to detect CFTR mRNA expression levels in the RSV persistent infected cells.3) Evaluate the changes of the functions of cystic fibrosis transmembrane transduction regulatory factor in human bronchial epithelial cells infected with RSV.Use whole cell recording method to record changes of RSV persistent infection in human bronchial epithelial cell model and negative control group and evaluate CFTR Cl-in model group cell. After the addition of agonist5μM forskolin, the activated cAMP could induce CFTR Cl-current, and then suppress cells at-40mv, followed by gradually increasing voltages from-100mv to+100mv with the schedule of polarization voltage at200ms and step order for20mv, interval of2s. Go back to the polarization voltage to40mv strangulation voltage. Take the maximum current value under each voltage state, draw i-v curve. Use MQAE fluorescent dye method to detect the cell chloride ion concentration of three groups of cells, prepare standard substance, detect standard substance chloride ion concentration, draw chloride ion concentration standard curve, calculate chloride ion concentration in measuring3groups bronchial epithelial cells according to the chloride ion concentration standard curve, and at the same time use MQAE fluorescent dye method to detect the cell chloride ion concentration fluorescence intensity change rate within three groups of cells.4) The change of epithelial repair function after human bronchial epithelial cells infected with RSVAfter preparing the damage model of RSV infecting human bronchial epithelial cells, use microscopic video to analyze BI-2000images/immunohistochemical analysis system to measure defect area respectively at0HRS,8HRS,12HRS,18HRS,24HRS after-mechanical damage, collect defected area images at all time points, draw the outlines of the edge of the defect areas, measure whether there is the existing linear correlation or not by using correlation coefficient, rendering time and repair area of the linear regression equation:Y=a+bX, calculate damage repair index (RI, repair index), RI value equals to the absolute value of b (RI=|b|) of the regression equation, to reflect the different repair speed of different treatment group, the slope of the straight line is damage repair index, the larger the slope is, the faster the damage repairs.Results:1) The establishment of RSV infected bronchial epithelial cells with airway hyper-responsiveness model.By using PCR method to validate RSV existence and its transcription expression level in the model, we found that after RSV being infected in the RSV infecting bronchial respiratory tract cell model, the RSV expression level of first few generations is increased gradually, and then maintains at a relatively stable level, but under the observation of the cell morphology, and no visible cell pathological changes can be found, also RSV is not removed by human bronchial epithelial cells. That is to say, under the RSV persistent infection, the establishment of infection model of bronchial epithelial cells with airway hyper-responsiveness is successful.2) By using immunofluorescence technique to detect the expression of CFTR in different group cells, we observed more green fluorescent expression of CFTR in the memcrane and cytoplasm of normal bronchial epithelial cells. And we observed less green fluorescent in epithelial cells infected by RSV. Compare with different generation cells infected by RSV, we observed less green fluorescent in stable infection bronchial epithelial cells with first generation cells. We inferred that after the cells was infected, its membranes and cytoplasm of visible more green dyeing CFTR. In RSV continuous model first generation infected cells, we found that the cell membranes and cytoplasm of CFTR were significantly reduced,. In RSV continuous model stability model group cell membranes and cytoplasm into negative control group in CFTR cell significantly reduced, pay RSV continuous model first generation cells also have a slight decrease, speculation RSV persistent infection cell, can destroy the integrity of the airway epithelium structure, which caused the unsteady state of local airway microenvironment, made the airway function disorder, changed the airway epithelium stress response mechanism, led to the remodeling of the airway function and structure, formed the change of chronic airway inflammation and airway pathologic.Extract negative control group cells of RSV persistent infection people bronchial epithelial cell model group respectively, membrane protein of model group cell of the first generation cells and stable RSV infection after RSV infection. Using Western-blot techique to detect the protein expression of RSV persistent infection cell CFTR. Process data of film exposure results by using Quantity one gray scanning software, its published strength showed with CFTR gray value/beta actin gray value, it repeated3times, counted results, drew histogram. The experimental results show that the CFTR expressions of the first generation cells after RSV infection and the model group cell of stable RSV persistent infection reduced significantly compared with negative control group cells. The difference is significant according to Student’s t test (*P<0.05).Extract negative control group cells in RSV persistent infection human bronchial epithelial cell model group respectively. After RSV infection the first generation cells and stable RSV infection model group cell RNA, reverse transcription cDNA, and use RT-PCR method to detect mRNA expression level of RSV persistent infection cells of CFTR. The result is mRNA expression=2(-△△C T) and shows that relative to the negative control group cells, after RSV infection the first generation cells and mRNA expression of model group cell CFTR of stable RSV persistent infection reduced significantly, and the results have obvious difference,*P<0.05.3) Functional changes of cystic fibrosis transmembrane transduction regulatory factor in human bronchial epithelial cells after being infected with RSV.Use whole cell recording method to record the changes of CFTR C1-in negative control group and model group cell of RSV persistently infected human bronchial epithelial cell model. The results showed that in RSV persistent infection model, CFTR C1-current density increased and depended on the concentration and maximum median effective dose was in10-3mM. CFTR C1-blockers, glibenclamide, can block the current. Rather than that, CFTR C1-blocker, DIDS, cannot block the current. MQAE fluorescent dye method can detect chloride ion concentration fluorescence intensity rate within the three groups of cells. The results showed that after RSV persistent infection, intracellular chloride ion concentration fluorescence intensity change rate was significantly reduced compared with the negative control group. It is indicated that after RSV persistent infection, channel protein regulating functions of regulating chloride ion channel are suppressed. And chloride ion outflow is reduced, intracellular chloride ion concentration rate was reduced.4) The changes of epithelial repair function after human bronchial epithelial cells were infected with RSV.The results show that the slope absolute value of RSV persistently infecting negative control group is bigger, repairing speed is increased. The slope absolute value of RSV persistently infecting model group is less, and the repairing speed is lowered. After RSV infection, its own proliferation of HBECs, migration and damage repair ability is reduced compared with RSV being infected previously, the damage repair function can remodel and have the potential to develop repeated attacks of airway inflammation and the reaction tendency.Conclusion:After human bronchial epithelial cells were infected by respiratory syncytial virus, the expression and function of CFTR on transmembrane were inhibited significantly. And the regulation and control function on the steady state of CFTR to airway epithelial cells were abated. Then, the local microenvironment of airway presents the loss of steady state. Affect the exocrine secretion of airway mucus from gland to airway. Form airway mucous plug, leading to increased in the airway resistance. Induce the happening and the development of airway responsieness disease.