A Study On The Effects Of Pressure Force On Airway Smooth Muscle Cells Via Co-cultured Epithelial Cells

Airway is the channel of aspiration and inspiration connecting throat and lung, and is mainly consisted of Airway epithelial cells, Fibroblast, Smooth muscle cells, Mast cells and so on. The normal respiration could be guaranteed by the rhythmic contraction of airway. Whereas, asthma can lead to the frequent convulsion of the airway wall and result in the scant of breath. What's worse, the repeat episodes of asthma will cause the great structural change of the airway wall, such as the proliferation and thickening of the airway smooth muscle, precipitation of the extracellular matrix etc., which will lead to the Airflow obstruction as well as the Non-specific Airway hyperresponsiveness.At present, asthma is still the worldwide difficulty which urgently needs to be solved. However, we haven't reached an agreement about the pathogenesis of asthma yet, so there are more basic researches to be done in order to deepen our understanding of asthma. Based on the asthma patients'symptoms like thickening of the airway smooth muscle and weakening of the contraction ability, the current study simulate the mechanical environment of body to investigate whether the biological features of normal airway smooth muscle cells and epithelial cells will be changed if they are exposed to external pressure stimulus.Epithelial cells are in the outermost layer of airway, under which are the smooth muscle cells. Epithelial cells can secrete some cytokines when they receive certain stimulation. Then these cytokines will make the smooth muscle cells change their biological features when they spread over the smooth muscle cells. In the current study, we first design a cell co-culture model and culture the epithelial cells and smooth muscle cells together; then we exert pressure to the system. Finally, the biological features of smooth muscle cells are examined to see if they are changed. The key researches and findings are as follows.①The primary culture and identification of epithelium cells and Smooth muscle cells of rat airway.We employ the method of tissue explant to culture smooth muscle cell of rat air duct, and then use the trypsin to digest the epithelium cells. After exploration of cell culture, tissue explant method can extract highly pure smooth muscle cells. In the sterile environments, we anatomise the rat to get the air duct, and then cut it into pieces. Next, these small parts of air duct are tiled in the cell culture bottle and cultured by DMEM/F12 culture medium for 6 days or so. Finally, we can get the target cells demonstrating the particular appearance of smooth muscle cell. With the help of tracheal perfusion, 0.05% trypsin can also digest more epithelium cells. After certain purification, we can get highly pure epithelium cells. When these two kinds of cells cover 80% of cell flask, we can employ 0.25% trpsin for digestion and cell passage.Then the two kinds of cells are identified. Firstly, in morphology, the two kinds of cells cater to the standard norm of each type respectively. Secondly, the specificα-actin of smooth muscle cells is tested by immuno-fluorescence staining, and we conclude that the kind of cell indeed is smooth muscle cells. Next, based on the epithelium cells'characteristic expression of Keratin as well as adopting cell immuno-histochemistry, it proves that we get the the epithelium cells successfully.②Proliferation of the Airway smooth muscle cell co-cultured with epithelial cell under physical forces.In this research, through MTT, we detect the proliferation of smooth muscle cell under 4 different culture circumstances, and draw the curve of growth for airway smooth muscle cell. The results indicate that comparing with other culture conditions, smooth muscle cells grow faster under the stimulation of pressure force and co-culture with epithelial cell. Through comparative analysis, it demonstrates that under physical forces epithelial cells will secrete certain cytokines, which can combine with smooth muscle cells and promote the proliferation of smooth muscle cells.③Stiffness of the Airway smooth muscle cell co-cultured with epithelial cells under physical forces.In the research,using Atomic force microscopy(AFM), we detect the young's modulus, that is the stiffness, of smooth muscle cells under four different cell culture environments. Through comparing, we draw conclusions as follows: comparing with other culture conditions, the co-culture with epithelial cell and pressure force increase the stiffness of smooth muscle cells significantly. It shows that through the virtual airway of co-culture system, the stiffness of smooth muscle cells were increased under the pressure force, it may match with the increase of asthma patients' potential contraction ability of airway.The results from this study all indicate that the co-culture of smooth muscle cells and epithelial cells can perfectly simulate the asthma patients'airway environment when they are exerted to pressure. And in this case, the smooth muscle cells are proliferated and stiffness becomes stronger. Thus, researchers can make use of this co-culture model in the further study. Specifically, through analyzing the cytokines in the culture solution, researchers can demonstrate which kind of cytokines is adopted by epithelial cells to affect the biological features of smooth muscle cells under pressure stimulus.