Construction Of A Simple Method For Isolation And Culture Of Primary Human Bronchial Epithelial Cells

BackgroundFrom a global perspective,in recent years,air pollution increased,especially haze pollution,respiratory system disease morbidity and mortality is also rising,in middleincome countries,the mortality of the top three diseases were cerebrovascular diseases(approximately 14.4% deaths),ischemic heart disease(ca.12.7%),chronic obstructive pulmonary disease(about 12%),especially in autumn and winter,respiratory disease morbidity and mortality is higher,the probability of human exposure to respirable particulate matter PM2.5 greatly increased the lung is the main factor.At present,China has become the world's most polluted areas,including Beijing,Tianjin,Hebei,the Yangtze River Delta,Chengdu,Chongqing and the Central Plains region is the world's most polluted areas,PM2.5 annual average air pollutants more than 70 micrograms per cubic meter,exceeding the national standard of more than 2 times higher than that of WHO.The guidance value of more than 7 times.From January 2009-2012 over the same period of satellite remote sensing data,January haze frequency and the affected scope and intensity showed an increase trend,especially in 2013 January the haze event spread over an area of 1 million 300 thousand square kilometers,with a population of 1 billion 100 million people affected by serious pollution haze,the worldwide,lasted for a long time,wide coverage,high levels of pollution are rare.This is a new requirement for the research and application of the pathogenesis,pathophysiology and drug toxicology of respiratory diseases.Research and training of primary bronchial epithelial cell separation for construction plays a very important role in the respiratory tract model,to further participate in the occurrence of respiratory diseases,the development of inflammation,drug reactions,air pollution control,public health and other aspects of research and application.At present,the separation of human primary bronchial epithelial cells has been reported by protease digestion and tissue block method,so that the samples obtained are dispersed into single cells for in vitro study.This study through fiberbronchoscope examination directly for individual bronchial epithelial cells without the need to go through protease digestion and tissue explant method for further separation,save the experimental steps and time,and as far as possible the original structure of primary bronchial epithelial cells retain many people,in order to explore a human bronchial epithelial cell in vitro culture and establish efficient and convenient the basic model.ObjectiveObjective to isolate,purify and optimize the primary culture methods of human bronchial epithelial cells in vitro,and identify them,so as to provide a good research carrier and experimental basis for the application of respiratory system related diseases and drug toxicology.Methods(1)In the bronchoscope room in a hospital of Xinxiang City,by the examination and consent of their families,access to relatively normal people by fiberbronchoscope examination of bronchial epithelial cells,the initial sampling,ice box and transported to the laboratory,to ensure that the higher cell survival rate of inoculated samples;(2)Purification,isolation and culture of human bronchial epithelial cells by serum-free BEGM medium.Primary culture and passage were performed.Trypan blue staining was used to detect cell viability.(3)The cell characteristics were observed and identified by inverted phase contrast microscope and immunohistochemical staining.(4)Using human interleukin 8(IL-8/CXCL8)ELISA kit and lactate dehydrogenase(LDH)kit to detect cell function.Results(1)A simple method of isolation of human primary bronchial epithelial cells can be established by fiberoptic bronchoscopy,and more individual bronchial epithelial cells can be obtained without the aid of digestive enzymes.(2)The survival rate of human primary bronchial epithelial cells obtained by brushing bronchoscopy is 96.8%.(3)The cell samples obtained by this method were cultured in the primary culture with the serum-free medium,and were collected and identified with the cytochemical staining of the specific marker keratin of the bronchial epithelial cells.It was proved that the cultured primary cells were human bronchial epithelial cells.(4)The human primary bronchial epithelial cells were obtained and cultured in vitro with different concentrations of inhalable lung particles PM2.5,and the levels of interleukin 8 and lactate dehydrogenase were detected in the cells after stimulation,and the primary bronchial epithelial cell work acquired and cultured by fiberoptic bronchoscopy was confirmed.It can be normal.Conclusions(1)It is a simple,easy to operate,quick and effective method to obtain and isolate human bronchial epithelial cells through brushing bronchoscopy.(2)The survival rate of human primary bronchial epithelial cells obtained by brushing bronchoscopy is 96.8%.(3)Primary cells obtained by brushing bronchoscopy proved to be human primary bronchial epithelial cells.(4)Human primary bronchial epithelial cells obtained by bronchoscopy were sensitive to PM2.5 and normal cell function.