Expression Of Cell Apoptosis And Correlative Signal In Epithelial Cells Of Idiopathic Pulmonary Fibrosis

Background and Objective Idiopithic Pulmonary fibrosis is a sort of disease with latent history, short course and high mortality. Few people have thoroughly investigated in it because of the limitation of medical treatment condition. Recently, along with the increasing environmental pollution and the enhancement of humanity contact with chemicals, the number of IPF patients is increasing year by year. In order to contribute to the early diagnosis and treatment, investigators hope to achieve breakthrough of the pathogenesis of IPF through the investigation of molecular biology. Investigations find that the molecular foundation of IPF was inflammatory reaction induced cell injury and fibrogenesis, but the clinical and experimental results of these investigations were not accord. So, investigation about apoptosis and its signal regulation in the pathogenesis of interstitial lung disease becomes a new hotspot. Recently, the expression and regulation of Fas(Apo-1/CD95)/Fas ligand (CD178) system which mediates death receptor pathway and p53 pathway in IPF are more concerned about. Cell apoptosis that is induced by activating effective caspases through the fas/fasL system is the major pathway of cell apoptosis. p53 is a key element which functioned "checkpoint", it contributes to DNA repair by blocking cell cycle at G₁ stage after cell DNA damage; if the damage of DNA can not be repaired, it could induce cell apoptosis. p53 is the most extensively investigated gene associated with apoptosis. At present, seldom investigation is concerned about the expression of Fas/FasL system in lung tissue of IPF patient. further more, there has no such a domestic research about the relationship of cell apoptosis, Fas/FasL system and P53 protein expression in IPF patient.The present study is designed in order to interpret the following points: to observe the cell apoptosis of IPF patient in bronchial and alveolar epithelial cells; to analyze the expression of Fas/FasL/P53 protein, Fas/FasL mRNA, and their correlation with cell apoptosis; to estimate correlation of P53 and Fas/FasL protein expression. We use terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL), immunohistochemistry and in situ hybridization to investigate the cell apoptosis, Fas/FasL/P53 protein, Fas/FasL mRNA expression and correlation of these factors in lung tissue of IPF patients, in order to investigate the role of them in the pathogenesis of IPF, and provide new theory foundation for the clinical diagnosis and therapy of IPF.Materials and methods 12 lung tissues were collected by open lung biopsies from patients with IPF. Ten patients are male, two are female. The diagnosis of IPF was based on strict criteria (ie, clinical history, physical examination, roentgenographic findings, laboratory tests, pulmonary function tests, histologic findings, and the exclusion of other known causes of interstitial lung disease), according to the latest American Thoracic Society/European Respiratory Society criteria. The histologic diagnosis in all specimens was compatible with that of UIP. Normal lung parenchyma specimens were obtained from 10 patients undergoing thoracotomy for isolated nodus. Patients in the control group had no other underlying lung pathology and 5 centimeters away from the nodus. The control group was comprised of 6 male subjects and 4 female subjects.Tissue samples were fixed in 10% formalin overnight and embedded in paraffin. A 4μm-thick paraffin section was adhered to slides which were treated by AEPS. Hematoxylin and eosin stain, TUNEL, immunohistochemistry and in situ hybridization were used respectively to examine the expression of cell apoptosis, p53/Fas/FasL protein and Fas/FasL messager RNA in bronchial and alveolar epithelial cells in lung specimens. The data were analyzed by SPSS 10.0, Mann-Whitney U-test were used to compare the percentage of IPF patients and control subjects with positive TUNEL results, immunohistochemical and in situ hybridization signals. The correlation between two markers, in patients and control subjects, was evaluated with the Spearman test using SPSS 10.0 too.α=0.05 was considered as level of test.Result1. The percentage of cell apoptosis detected by TUNEL in alveolar and bronchial epithelial cells of IPF patients was 100.0% (12/12), it was higher than control group's 0.0% (0/10), the difference was significant (U=0.000, P<0.01).2. Fas mRNA stained positive in alveolar epithelial cells and bronchial cells in all IPF patients, the positive percentage was 100.0% (12/12), in control subjects, only 30.0% (3/10) (U=5.500,12.500, P<0.01); FasL mRNA stained positive in alveolar epithelial cells and bronchial cells in IPF patients too, the percentage was 75.0% (9/12), but it stained 90.0% positive in control subjects (9/10), it was no statistical difference between two groups (U=51.000, P > 0.05).3. Fas protein stained positive in alveolar epithelial cells in all IPF patients, the positive percentage was 100.0%, (12/12), and in few alveolar epithelial cells in 50.0% of control subjects (5/10) (U=12.0, P<0.01); bronchial cells were stained positive by Fas protein in all IPF patients, the percentage is 100.0% (12/12), which were significantly higher than control group, the positive percentage was 60%, (6/10) (U=24.0, P<0.01); FasL protein stained positive in alveolar epithelial cells and bronchial cells in all IPF patients too, percentage 100.0%, (12/12), there were stained positive 20.0% in alveolar epithelial cells(2/10)/30.0% in bronchial cells(3/10)of control subjects (U=7.0,8.0, P<0.01). Compared with control group's positive percentage 0.0% (0/10), positive stained P53 protein signals of alveolar epithelial cells and bronchial cells in IPF patients were significantly higher, the percentage was 91.7%, (11/12) (U=5.0,P<0.01).4. There was signifcant correlation between TUNEL results and the expression of Fas/FasL/P53 protein, Fas mRNA(r_s=0.625～0.839, P<0.01). There was no correlation between TUNEL results and FasL mRNA (r_s=-0.078～-0.033, P > 0.05). There was significant correlation between the expression of Fas/FasL and P53 protein (r_s=0.571～0.760, P<0.01).Conclusion1. Compared with the control group, Cell apoptosis in alveolar epithelial cells and bronchial cells of IPF patients is significantly higher, cell apoptosis could contribute to fibrogenesis, it maybe one of the key elements in pathogenesis of IPF.2. Fas/FasL/P53 protein,Fas mRNA expression is higher in alveolar epithelial cells and bronchial cells of IPF patients, and there is significantly statistical correlation between TUNEL results and the expression of these marks.3. There is significant statistical correlation between Fas / FasL and P53 protein expression in alveolar epithelial cells and bronchial cells, Which indicates Fas/FasL and p53 cooperate on these cells and make their apoptosis ascending.