The Primary Culture And Identification Of Fibroblasts Cells From Nasal Polyps Between Normoxia And Hypoxia

Objective:To investigate the primary culture and identification of fibroblasts cells from nasal polyps between normoxia and hypoxia for further study on their biological features.Methods:The specimens of nasal polyps(20cases)endoscopically resected during in October2012to March2013on Shandong university qilu hospital otolaryngology-head and neck surgery were placed into4℃sterile phosphate-buffered saline(PBS)soIution supplemented with penicillin and streptomycin, washed repeatedly, immersed, cut into pieces, tube into two portions to EP, respectively with1ml of0.1%collagenase I and1ml of dispase II overnight. The next day, the samples were removed, pipetted slightly to detach the epithelia, after to count, added in broth (DMEM+10%fetal bovine serum+1×10U/L penicillin+100mg/L streptomycin), respectively in normoxia and hypoxia conditions,(normoxia training with air as the mixed gas, oxygen volume fraction of about20%; hypoxia training with nitrogen as the mixed gas, regulating oxygen volume fraction of5%. The other culture conditions are same with the volume fraction of5%CO2and saturated humidity at37℃) Under an inverted phase microscope, the cells morphology, proliferation, growth and infection were observed. And nasal polyp morphology preliminary appraisal of fibroblasts, with vimentin monoclonal antibody immunohistochemical staining for identification. The proliferation of cells were tested by CCK-8, and to describe cell growth curve. Results:Overall improvement was observed in the number of cells in dispase II and that of type collagenase I (P<0.05). It can be isolated from nasal polyp tissues fibroblasts, and to develop, under the inverted phase contrast microscope to observe the morphology of the cell into fusiform, elongated, polygonal or irregular shape. The central ovoid nuclei, cytoplasm2-3protruding from the length of irregular protrusions, high cell density, cell parallel arrangement or radiated and vortex pattern. Waveform of fibroblasts monoclonal antibody immunohistochemical staining, immunohistochemical staining after a brown or brown markers for vimentin positive cells, confirmed as fibroblasts. Nasal polyp fibroblasts in normoxia and hypoxia condition training have no obvious difference.Conclusion:Dispase II can be used to obtain more cells. In normoxia and hypoxia condition, the above mentioned model is more suitable for primary culture due to its faster proliferation, and more stable and reliable cell supply for nasal polyps research..