| Chronic obstructive pulmonary disease(COPD)is the third leading cause of death worldwide due to its high morbidity and mortality.Long-term cigarette smoking is the main cause of COPD.In addition to cigarette smoking,long-term inhalation of biofuel smoke and environmental pollution particles can also cause COPD.COPD is characterized by airway remodeling and emphysema.Airway remodeling was caused by abnormal proliferation of airway basal cells,leading to thickening of the airway inner layer and narrowing of the airway cavity.Airway remodeling was also caused by abnormal differentiation of airway basal cells,showing squamous cell metaplasia,ciliated cells decreasing,goblet cell and submucosal glands hyperplasia,which induces increased mucus secretion and decreased ciliary clearance,leading to the impaired mucociliary clearance(MCC)function.The reduction of ciliated cells,the reduction and shortening of cilia,as well as the improper ciliary beating frequency can lead to dysfunction of cilia,which accounts for a large proportion of impaired MCC.COPD is characterized by chronic inflammation,infiltration of neutrophils,macrophages,and lymphocytes,with the largest number of macrophages.Macrophages are important guardians of lung homeostasis.In addition to phagocytosis and clearance of inhaled particulate pathogens,presentation of antigens,and production of cytokines to initiate adaptive immunity,they also play an indispensable role in tissue repair and maintenance of tissue homeostasis.Activated macrophages secrete many chemokines and cytokines,such as CXCL8 which attracts neutrophils and monocytes into the lung,matrix metalloprotenase(MMP)which cause lung tissue destruction to develop emphysema,and TGF-?which participate in tissue repair and induce tissue remodeling.These chemokines and cytokines play an important role in the development of COPD.In our experiment,we found that macrophages secreted more Bone morphogenetic protein(BMP)-2 after stimulation with cigarette smoke extract.BMP-2 belongs to the TGF-?superfamily.BMP signaling pathway is highly conserved from flies to mammals and plays an important role in the development of multiple organs.BMP signal has an effect on the renewal and differentiation of pluripotent stem cells.For example,the deficiency of BMP signal in the process of lung development would lead to bronchopulmonary dysplasia,and the enhancement of BMP-2 signal in rat neuroepithelial cells could inhibit the differentiation of neurons.After airway epithelial injury,basal cells which have stem cell characteristics could replenish the injured epithelial cells through proliferation and differentiation.Therefore,it is speculated that BMP-2 might affect airway epithelial cell differentiation through some mechanism.By means of acute or chronic cigarette exposure for mice and two cell lines co-culture system,this experiment focus on the effect of macrophages on the differentiation of airway epithelial cells,and especially focus on the expression of ciliary protein of the epithelial cells and on the role of BMP-2plays in this process.It will indirectly show us the role of macrophages on the differentiation of airway basal cell in the development of COPD.Part one Effects of acute and chronic cigarette smoke exposure on the airway epithelial differentiation and inflammatory cell infiltration in the lung of miceObjective:The effects of acute and chronic cigarette exposure on the airway epithelial differentiation and inflammatory cell infiltration in the lung of mice were observed.Methods:1.Mice were exposed acutely to high doses of cigarette smoke for one month.The RNA and proteins are extracted form the trachea and the lung of mice.The expression of Foxj1m RNA and beta-tubulin-?protein of ciliated cells in trachea and the expression of muc5ac m RNA and muc5ac protein as well as scgb1a1m RNA and CCSP protein of secretory cells in the lung was detected by RT-PCR and WB tests;Single cell suspension was prepared from fresh lung tissue of mice,incubated with fluorescence antibody,and the proportion of CD11b~+F4/80~+macrophages,CD3~+CD4~+T cells,CD3~+CD8~+T cells,CD4~+CD25~+Fox P3~+Treg cells was detected by FACS.The numbers of CD68 positive macrophages was detected by IHC staining in lung tissue sections of mice.2.Mice were exposed chronically to cigarette smoke for four months,COPD model was confirmed.The RNA and proteins are extracted form the trachea and the lung of mice.The expression of Foxj1m RNA and beta-tubulin-?protein of ciliated cells and the scgb1a1 m RNA of secretory cells in the trachea and the expression of muc5ac m RNA and muc5ac protein in the lung was detected by RT-PCR and WB tests.The expression of beta-tubulin-?protein and CCSP protein was detected by IF staining in the trachea tissue section of mice.Single cell suspension was prepared from fresh lung tissue,incubated with fluorescence antibody,and the proportion of CD11b~+F4/80~+macrophages,CD3~+CD4~+T cells,CD3~+CD8~+T cells,CD4~+CD25~+Fox P3~+Treg cells was detected by FACS.The numbers of CD68positive macrophages was detected by IHC staining in lung tissue sections.Results:1 The effects of acute cigarette smoke exposure on airway epithelial differentiation and inflammatory cells of mice.1.1 The effects of acute cigarette smoke exposure on the inflammatory cells of mice.1.1.1 Acute cigarette smoke exposure led to the increase of macrophages in the lung of mice.After acute cigarette smoke exposure for one month,the CD11b~+F4/80~+macrophages in the lung detected by FACS and the CD68~+macrophages in the lung detected by IHC staining were increased significantly in the CS group(P<0.05).1.1.2 Acute cigarette smoke exposure led to the increase of CD3~+CD4~+T lymphocytes in the lung of mice.After acute cigarette smoke exposure for one month,the percentage of CD3~+CD4~+T lymphocytes in the lung detected by FACS were increased significantly in the CS group(P<0.05).1.2 The effects of acute cigarette exposure on the airway epithelial differentiation of mice.1.2.1 Acute cigarette smoke exposure led to the decrease of ciliated cells in the trachea of mice.After acute cigarette smoke exposure for one month,the expression of foxj1 m RNA and beta-tubulin-?protein in the trachea was decreased significantly in the CS group(P<0.05).1.2.2 Acute cigarette smoke exposure led to the increase of mucus cells in the lung of mice.After acute cigarette smoke exposure for one month,the expression of muc5ac m RNA and muc5ac protein in the lung was increased significantly in the CS group(P<0.05).1.2.3 There is no apparent changes of Clara cells in the lung of mice after cigarette smoke exposure for one month.After acute cigarette smoke exposure for one month,the expression of scgb1a1 m RNA and CCSP protein in the lung was not changed obviously.2 The effects of long-term chronic cigarette smoke exposure on airway epithelial differentiation and inflammatory cells of mice.2.1 The effects of long-term chronic cigarette smoke exposure on the inflammatory cells of mice.2.1.1 Chronic cigarette smoke exposure led to increased macrophages in the lung of mice.After chronic cigarette smoke exposure for four months,the CD68~+macrophages in the lung detected by IHC staining were increased significantly in the CS group(P<0.05).2.1.2 Chronic cigarette smoke exposure led to the decrease of lymphocytes in the lung of mice.After chronic cigarette smoke exposure for four months,the percentage of CD3~+CD4~+T,CD3~+CD8~+T and the CD4~+CD25~+Fox P3~+Treg lymphocytes in the lung detected by FACS was decreased significantly in the CS group(P<0.05).2.2 The effects of long-term chronic cigarette smoke exposure on the airway epithelial differentiation of mice.2.2.1 Chronic cigarette smoke exposure led to the decrease of ciliated cells in the trachea of mice.After chronic cigarette smoke exposure for four months,the expression of foxj1 m RNA and beta-tubulin-?protein in the trachea was decreased significantly in the CS group(P<0.05).The ratio of beta-tubulin-?protein positive cells detected by IF staining in section of the trachea was decreased significantly in the CS group(P<0.05)2.2.2 Chronic cigarette smoke exposure led to the increase of mucus cells in the lung of mice.After chronic cigarette smoke exposure for four months,the expression of muc5ac m RNA and muc5ac protein in the lung was increased significantly in the CS group(P<0.05).2.2.3 Chronic cigarette smoke exposure led to the increase of Clara cells in the trachea of mice.After chronic cigarette smoke exposure for four months,the expression of scgb1a1 m RNA and CCSP+protein cells in the trachea was increased significantly in the CS group(P<0.05).Summary:1.Acute cigarette exposure in mice led to decreased ciliated cells in the trachea and increased mucus cells in lung.At the same time macrophages and CD3+CD4+T cells in lung tissues were increased.2.Long-term chronic cigarette smoke exposure in mice led to decreased ciliated cells,increased Clara cells in the trachea and increased mucus cells in the lung.Macrophages were increased but CD3+CD4+T,CD3+CD8+T,CD4+CD25+Fox P3+Treg cells were reduced significantly in the lung of mice.Part two The Effects of macrophages on the ciliary protein expression of airway epithelial cells after cigarette smoke extract stimulationObjective:The effects of cigarette smoke extract and macrophages on the ciliary protein expression of airway epithelial cells was observed in co-culuted system.The screen test was carried out to find out the cytokines secreted by macrophages which have an effect on the ciliary protein expression through high-throughput sequence test on RNA of macrophages.Methods:1.Different concentrations of CSE were added into the medium to observe the effect of CSE on beta-tubulin-?protein expression of Beas-2B cells.2.The effects of Thp-1 cells on beta-tubulin-?protein expression of Beas-2B cells was observed when Thp-1 cells and Beas-2B cells were co-cultured.3.High-throughput sequence test was performed on the m RNA of Thp-1cells in different conditions to screen out cytokines that might have an effect on beta-tubulin-?protein expression of Beas-2B cells.Results:1 The effects of different concentrations of CSE on the ciliary protein expression of Beas-2B cells were different.0.25%CSE could increase the expression of ciliary protein of Beas-2B cells.However,1%CSE has the inverse effect.2 The effect of Thp-1 cells on the ciliary protein expression of Beas-2B cells.2.1 The effect of Thp-1 cells on the expression of ciliary protein of Beas-2B cells with or without CSE stimulation was different.When Thp-1 cells and Beas-2B cells were co-cultured in transwell,Thp-1 cells could inhibit the ciliary protein expression of Beas-2B cells.When CSE was added into the co-culture system,the inhibitory effect was augmented.2.2 The inhibitory effect of Thp-1 cells on the ciliary protein expression of Beas-2B cells was concentration dependent.When Beas-2B cells were co-cultured with Thp-1 cells under CSE stimulation,Thp-1 cells inhibited the expression of ciliary protein of Beas-2B cells.This inhibitory effect was further enhanced with the increased numbers of Thp-1 cells.3 BMP-2 was screen out to be considered to have an effect on ciliary protein expression of Beas-2B cells.Summary:1.0.25%cigarette smoke extract could increase the expression of ciliary protein of Beas-2B cells.While 1%cigarette smoke extract did the opposite.2.Thp-1 cells could inhibit the expression of ciliary protein of Beas-2B cells,which was enhanced by cigarette smoke extract stimulation.3.BMP-2 was screen out to be considered to have effect on ciliary protein expression of Beas-2B cells.Part three The Effect of BMP-2 on the ciliary protein expression of airway epithelial cells after cigarette smoke extract stimulationObjective:The effects of recombinant human BMP-2 protein,Thp-1cells,and the BMP-2 si RNA on the expression of ciliary protein of Beas-2B cells were observed when the recombinant human BMP-2 protein and or the BMP receptor inhibitor LDN was added into the culture system or when Thp-1cells were interfered with BMP-2 si RNA.Methods:1.The effects of different concentrations of recombinant human BMP-2protein on the expression of ciliary protein of Beas-2B cells were observed when recombinant human BMP-2 protein and CSE were added into the medium.2.The effects of recombinant human BMP-2 protein on the expression of ciliary protein of Beas-2B cells were observed when recombinant human BMP-2 protein was into the medium with or without the stimulation of CSE.3.The effects of BMP receptor blockage on the expression of ciliary protein of Beas-2B cells were observed when Beas-2B cells were pretreated with LDN for 24 hours before recombinant human BMP-2 protein and CSE were added into medium.4.The effects of BMP receptor blockage on the expression of ciliary protein of Beas-2B cells were observed when Beas-2B cells were pretreated with LDN for 24 hours before co-cultured with Thp-1 cells with CSE stimulation.5.The effects of Thp-1 cells on the expression of ciliary protein of Beas-2B cells when Thp-1 cell was pretreated with BMP-2 si RNA for 24hours before co-cultured with Beas-2B cells with CSE stimulation.Results:1 The effect of recombinant human BMP-2 protein on the expression of ciliary protein of Beas-2B cells1.1 Recombinant human BMP-2 protein has an inhibitory effect on the expression of ciliary protein of Beas-2B cells in a concentration dependent manner with the stimulation of CSE.Recombinant human BMP-2 protein could decrease the expression of ciliary protein of Beas-2B cells with the CSE stimulation.The inhibitory effect was augmented by higher concentration of recombinant human BMP-2 protein.1.2.The effect of recombinant human BMP-2 protein on the expression of ciliary protein of Beas-2B cells was different with or without the stimulation of CSE.Recombinant human BMP-2 protein has no apparent effect on the expression of ciliary protein of Beas-2B cells without CSE stimulation.However,recombinant human BMP-2 protein could decrease the ciliary protein expression of Beas-2B cells significantly with the CSE stimulation.2 The effect of Thp-1 cells on the ciliary protein expression of Beas-2B cells after BMP signal was inhibited.2.1 The inhibitory effect of recombinant human BMP-2 protein on the ciliary protein expression of Beas-2B cells could be blocked by BMP receptor blockage LDN.Recombinant human BMP-2 protein could decrease the expression of ciliary protein of Beas-2B cells with the CSE stimulation.This inhibitory effect could be blocked when Beas-2B cells were pretreated with LDN for 24 hour before co-cultured with Thp-1 cells and with the stimulation of CSE.2.2.The inhibitory effect of Thp-1 cells on the ciliary protein expression of Beas-2B cells can be partly blocked by BMP receptor blockage LDN.Thp-1cells could inhibit the expression of ciliary protein of Beas-2B cells when co-cultured with Beas-2B cells and with the stimulation of CSE.However,this inhibitory effect can be partly blocked when Beas-2B cells were pretreated with LDN for 24 hours before co-cultured with Thp-1 cells.2.3 BMP-2 si RNA could partly block the inhibitory effect of Thp-1 cells on the ciliary protein expression of Beas-2B cells with the stimulation of CSE.Thp-1 cells have an inhibitory effect on the expression of ciliary protein of Beas-2B cells when were co-cultured with Beas-2B cells and with the stimulation of CSE.But when Thp-1 cells were pretreated with BMP-2 si RNA for 24 hours before co-cultured with Beas-2B cells,the inhibitory effect of Thp-1 cells on the ciliary protein expression can be partly blocked.Summary:1.The recombinant human BMP-2 protein could decrease the ciliary protein expression of Beas-2B cells in a concentration dependent manner with the stimulation of CSE.2.The inhibitory effect of Thp-1 cells on the expression of ciliary protein of Beas-2B cells could be partly blocked when Beas-2B cells were pretreated with BMP receptor inhibitor LDN or when Thp-1 cells were interfered with BMP-2 si RNA before co-cultured.Conclusion:1.Acute or chronic cigarette smoke exposure would lead to the decrease of ciliate cells in the trachea and the increase of mucus cells in the lung.At the same time,it would cause the increase of macrophages in the lung.2.0.25%cigarette smoke extract would increase the ciliary protein expression of airway epithelial cells,while 1%cigarette smoke extract has the opposite effect.3.Macrophages could inhibit the ciliary protein expression of airway epithelial cells.Which was augmented by cigarette smoke extract stimulation.4.BMP signal inhibition can partially block the inhibitory effect of macrophages on the ciliary protein expression of airway epithelial cells.5.Macrophages inhibit the ciliary protein expression of airway epithelial cells through BMP-2 secreted from macrophages after cigarette smoke extract stimulation. |
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