The Study On The Biological Effects Of Cyclic Substrate Strain On Human Pulmonary Epithelial Cells

The biological effects of cyclic stretching on the human pulmonary epithelial cells were studied through biomechanical, cell biological, biochemical and immunofluorescence methods and technology. The mechanism by which human pulmonary epithelial cells respond to mechanical signal was investigated at cellular and molecular levels in the present paper. The main work and conclusion as follow:(1) An model in vitro for human pulmonary epithelial cells was set up and used for investigating the effects of mechanical strain on the pulmonary epithelial cell. Its feasibility was studied by biochemisrty and flow cytometry.(2) To determine the proliferative effect of mechanical strain on human pulmonary epithelial cells, cell cycle analysis was carried out by flow cytometry. The results showed that the A549 and H727 cellular proliferative index was inhibited. A549 cellular proliferative index was not significantly different (P>0.05) after 24 h of cyclic strain at frequency 20 cycles/min or 40 cycles/min. However, cyclic strain caused a significant reduction in the A549 cellular proliferative index of 48 h at frequency 20 cycles/min (P<0.05), and at frequency 40 cycles/min (P<0.01). Similarly, cyclic strain caused a significant reduction in the H727 cellular proliferative index of 24 h, 48 h, 72 h at frequency 20 cycles/min and at frequency 40 cycles/min (P<0.01). There were statistical significance (3) To test the hypothesis that mechanical strain activates integrins by causing its clustering, A549 and H727 cells were kept static or subjected to 15% elongation at frequency 40 cycles/min for 24 h followed by anti-α3, anti-α5 and anti-β1 integrin immunostaining. The reorganization of cell surface integrins was observed by confocal microscopy. The results revealed that α3 integrin,α5 integrin and β1 integrin in A549 and H727 were more concentrated and brighter, and cells created a large fusion of focal adhesion or adhesion plaques after 24 h exposure to strain.Under static condition, α3 integrin,α5 integrin and β1 integrin in A549 cells lacalized in random speckle pattern resembling focal adhesions and the fluorescence intensity was weak, but α3 integrin,α5 integrin and β1 integrin in H727 cells maintained a diffusepattern with an occasional speckle concentration in some regions. Furthermore α3 integrin, α5 integrin and β1 integrin transferred to cellular basal layer and created a large fusion of focal adhesion, and affinity between integrins and ECM was enhanced.(4) The micropipette aspiration was used to study the effect of cyclic strain on mechanical property of human pulmonary epithelial A549 and H727 cells. The cyclic strain increased cells adhesion foreces and spreading areas. Furthermore, A549 and H727 cells adhesion foreces and spreading areas increased with the loading time.The results showed that human pulmonary epithelial A549 and H727 cells responded to mechanical strain by changing their mechanical patterns.(5) A549 and H727 cells were stained with rhodamine-phalloidin, confocal microscopy revealed that cyclic strain promoted the polymerization of actin monomers into the stress fiber. A intact structural sequence is formed between substratum, integrins and cytoskeleton. The structural sequence can play a pivotal role in regulating growth, migration,adhesion of human pulmonary epithelial cells, and activation of some signaling such as protein tyrosine kinases, ion channels i.e. at focal adhesion sites.(6) The intracellular calcium plays an important role in cell mechanotransduction.The fluorescence probe was used to investigate the changes of intracellular calcium concentration when human pulmonary epithelial cells responded to stretching. The results showed that the rise of intracellular calcium concentration was due to the influx of extracellular calcium and the release of intracellular calcium store ,in which the influx of extracellular calcium play the main role.