Study On HIF And The Proliferation Rate Of Human Tracheal Fibroblasts

Backgroud:Tracheal intubation and ventilator-assisted ventilation is an important mean to rescue patients and the guarantee of operation.However,there are many complications,for example,ventilator-associated pneumonia,barotrauma,etc,and there are also so many long-term complications,including tracheal stenosis,trachea-esophageal fistula.In tracheal intubation,the proliferation of tracheal mucosal cells results in tracheal stenosis,while the apoptotic reaction results in tracheoesophageal fistula,and it is generally acknowledged that the primary factor of the series of mucosal reactions is hypoxia.Besides,in a study,hypoxia inducible factor(HIF)was found in patients with tracheal stenosis after endotracheal intubation.Hypoxia inducible factor(HIF)is expressed in cells and it regulates the expression of a series of genes to maintain the homeostasis organisms in hypoxic environment,and the most of responses to hypoxia are regulated by HIF-1α and HIF-2α.HIF-la and HIF-2α play a self-contradictory role in cell proliferation and apoptosis,they not only induce cell apoptosis,but also prevent cell apoptosis and even promote cell proliferation.Therefore,we speculate that tracheal stenosis and tracheoesophageal fistula after endotracheal intubation would have two different outcomes under hypoxic stress by influencing the proliferation and apoptosis of tracheal mucosal cells with HIF.Objective:In this study,human tracheal fibroblasts were cultured under different degrees of hypoxia.It was verified that human tracheal fibroblasts were based on HIF and affected the proliferation/apoptosis of human tracheal fibroblasts by regulating the expression of HIF-la/HIF-2a,Thus,tracheal stenosis/tracheal-esophageal fistula after tracheal intubation was confirmed to be tracheal mucosa under different degrees of hypoxia from the cytokine level.With HIF as the core,by regulating the expression of HIF-1α/HIF-2α,two outcomes are produced,which provides a new idea and target for the prevention and treatment of tracheoesophageal fistula/tracheal stenosis.Methods:Human tracheal fibroblasts were divided into four groups according to the oxygen concentration in incubator:21%O2(1st group),10%O2(2nd group),5%O2(3rd group)and 1%O2(4th group),the cell proliferation rate of each group was detected by CCK-8 kit,Real-time fluorescent quantitative PCR(RT-PCR)and Western blot analysis were employed to respectively detect the mRNA transcription and protein expression levels of HIF-1α,HIF-2α,HIF-1α/HIF-2α.Results:1.Comparison of proliferation rate of human fibroblasts in different oxygen concentration:Compared with the 1st group,the proliferation rate of human fibroblasts was higher in the 2nd group,while the proliferation rate of human fibroblasts was lower in the 4th group.2.The mRNA level of HIF-la and HIF-2α in human fibroblasts at different oxygen concentration:In the 1st group,the mRNA level of HIF-1α was significantly higher than the 2nd group,while the mRNA level of HIF-1α was significantly higher in the 3rd group and the 4th group than the 1st group.In the 4th group,the mRNA level of HIF-2α was higher than the others,while there were not different among the 1st group,3rd group and the 4th group.3.The protein level of HIF-la and HIF-2α protein in human fibroblasts at different oxygen concentration:In the 2nd group,the protein level of HIF-1α was significantly lower than the 1st group,while the protein level of HIF-1α was significantly higher in the 3rd group and the 4th group than the 1st group.In the 1st group,the protein level of HIF-2α was lower than the others,and the protein level of HIF-2α in the 3rd group was highest.4.Analysis of the correlation between the protein level of HIF-1α/HIF-2α and the proliferation proliferation rate of human tracheal fibroblasts:The protein level of HIF-1α/HIF-2α protein was linearly correlated with the proliferation rate of human tracheal fibroblasts(P=0.005),and the intensity of the relationship was-0.870,which was negatively correlated.Conclusion:The ratio of HIF-la/HIF-2α protein affects the proliferation rate of tracheal fibroblasts at different oxygen.In hypoxic condition of 10%O2,the protein level of HIF-1α/HIF-2α decreased,which accelerated the proliferation rate of human tracheal fibroblasts.However,in severe hypoxic condition of 1%O2,the protein level of HIF-1α/HIF-2α decreased,which slowed down the proliferation rate of human tracheal fibroblasts.