| As a chronic respiratory disease, asthma is becoming an increasingly serious threatto human health. Unfortunately, the etiology and development of asthma are still notwell understood, while the pathological mechanism of asthma remains difficult toelucidate.The airway are tubes that carry air into and out of your lungs. People who haveasthma have inflamed airways,This makes the airways swollen and very sensitive.When the airways react, the muscles around them tighten. This narrows the airways,causing less air to flow into the lungs. The swelling also can worsen, making theairways even narrower. Cells in the airways may make more mucus than normal. Mucusis a sticky, thick liquid that can further narrow your airways.Airway smooth muscle proliferation is one of the onset of symptoms of asthma.Ithas been reported that cultured airway myocytes from subjects with asthma proliferatefaster than do those from non-asthmatic individuals.Although corticosteroids inhibitproliferation of normal airway myocytes by reducing cyclin D1expression andretinoblastoma protein phosphorylation), they apparently fail to inhibit proliferation ofairway myocytes from subjects with asthma, Despite done so much research, manyproblem are unknown.Airway epithelial cells play a critical role in the defense system of the lungs byproviding an important barrier function to potentially toxic environmental agents thatcan promote epithelial damage, epithelial shedding,marked by increased numbersofepithelial cell clumps (creolabodies) in sputum and bronchial epitheliumdesquamation, are features of airway injury. The sequelaeis an airwaye pithelium in achronic state of wound repair, which secretes soluble mediators necessary for cellproliferation, migration, and extracellular matrix synthes is consistent with a healingwound environment.Both airway smooth muscle and airway epithelial cells have significantpathological features in asthma, but is there signal exchange between them and howthey exchange singnal is still unknow until now. In this project, We established injuriedepithelial cell and normal smooth muscle cell co-culture model in vitro. In asthma,eosinophils are major effector cells, eosinophil cationic protein (ECP) is one of maineffect compositions. Poly-L-arginine (poly-L, of arginine, PLA), as the analogue of the ECP, has been widely used in the experimental study of asthma. We exert externalpressure on the airway epithelial cells co-cultured with airway smooth muscle cells tosimulate the enviroment of airway epithelial tissue more authentically in asthma. Thenwe detected the proliferation of ASMC to make a preliminary exploration of influenceof airway epithelial cells on airway smooth muscle tissue when asthma happened.①In order to set up the model in vitro: this research collected and cultured airwaysmooth muscle cells and airway epithelial cells in vitro, identifying the primary cell bymorphology and immunofluorescence. The results showed that we obtained high purityprimary airway smooth muscle cells by tissue adherent and airway epithelial cells bycold digestion with trypsin.②The research on PLA affect rat airway epithelial cells activity: set PLAconcentration as0-50μM (every5μM as an interval), total ten concentration gradients,count the number of living cell by trypan blue dying. The final results showed,compared with the control group, even if the lowest concentration of PLA (5μM) hadapparent toxic effects on airway epithelial cells (P <0.05). And when the concentrationgreater than20μM, the toxic effects were no longer obvious change.③Set up airway epithelial cell injury model: treated the airway epithelia cell for ashort period of time (10minutes) using a high concentration of PLA (50μM), build theinjure model.④Design the co-culture device: in order to put pressure on the airway epithelialcells alone when in co-cultured, we improved the pressure device of co-culture. Finally,the developed device can provide the stability of the pressure we need (14KPa).⑤In this study, we set up five experimental groups:cultured alone for the controlgroup (A),ASMC and AEC culture (B), ASMC and injured AEC culture (C), ASMC andinjured AECculture (D), ASMCco-cultured with injured AEC and exert externalpressure (E)⑥detection of smooth muscle cells in the condition of co-culture：Measuring theOD value of an airway smooth muscle cell growth by MTT every12h, and take atotal of five time points, both12h,24h,36h,48h,60h, respectively, andthe results show that:60h, the p value of BCDE contrast with A <0.01, ASMCgrowth rate of group E was significantly higher than other groups. and Group D andGroup E and Group C these is significant differences between Group E with GroupC and Group D(p<0.01), we can infer that external pressure and AEC injuryindependent of the role of value- added on the ASMC。We finally concluded that, in vitro studies, airway epithelial damage and acceptan external stimulus. Can significantly improve the airway smooth muscle proliferation. |
PDF Full Text Request