| Idiopathic pulmonary fibrosis is a chronic progressive fibrosis interstitial pneumonia characterized by progressive dyspnea,decreased exercise endurance,interstitial infiltration of the lung parenchyma,and pulmonary restrictive pulmonary ventilation dysfunction.Respiratory failure is the fundamental cause of death in IPF patients.The mean median survival period after diagnosis is 3-5 years.There is no effective treatment except for lung transplantation.The disease is mainly the disease of the elderly,and occurs mostly in adult men.The age of diagnosis is more than 65 years old.At present,there is evidence that there is a correlation between IPF and aging.It is an aging related disease with various causes and ways.Telomerase and DNA methylation are all related factors of cell aging and participate in the development of IPF.In the development of IPF,TGF-beta can cause telomerase abnormality,and DNA methylation may participate in it.TGF-beta and telomerase as important molecules are involved in the process of IPF,which may make cells continue to produce a vicious cycle of cell replication.Studies have shown that TERT and DNMT3 a and DNMT3 b are considered to be the key speed limiting factors for telomerase activity and DNA methylation respectively.It can be used to study telomerase and DNA methylation as an indicator of telomerase activity and the level of DNA methylation,respectively.Can TGF-beta1 lead to changes in telomerase activity and DNA methyltransferase in IPF? How does it change?Are there any related interventions?This project will focus on the changes of telomerase and DNA methyltransferase and the relationship between telomerase and DNA methyltransferase in the study of TGF-beta1,and explore its role in the pathogenesis of IPF.First,the expression of TERT,DNMT3a,DNMT3 b and TGF-beta1 in peripheral blood of IPF patients and normal population was studied.The expression of TERT,DNMT3 a,DNMT3b and TGF-beta1 in IPF patients and normal population was different,and the expression of TERT,DNMT3 a,DNMT3b and TGF-beta1 in pulmonary fibrosis mice was further studied in animal experiments.To study the abnormal expression,interaction and intervention measures of TERT and DNMT3 a and DNMT3 b induced by TGF-beta 1 in A549 human alveolar type II epithelial cells.It is confirmed that TGF-beta1 regulates the expression of TERT through DNA methylation and puts forward relevant intervention measures.It is divided into three parts and six experiments.Part one: The expression of TERT,DNMT3 a,DNMT3b and TGF-beta1 in peripheral blood of IPF patientsExperiment one: The expression of TERT,DNMT3 a,DNMT3b and TGF-beta1 in peripheral blood of IPF patientsObjective:To research the expression and significance of Peripheral blood leukocyte TERT,DNMT3 a and DNMT3 b in peripheral blood serum from peripheral blood of IPF patients.Methods:Choose ten male IPF patients 60 years of age or older not associated with other disease as the experimental group,choose healthy ten male volunteers 60 years of age or older as control group.Collect basic clinical datas,The effect of ELISA on the expression of serum TGF-beta1,IL-1beta,IL-6 and DNMT3 a and DNMT3 b in the control group and IPF group;the effect of Western Blot method on the expression of TERT protein in peripheral blood leukocytes in control group and IPF group.Results:1.The expression of TGF-beta1,IL-1beta,IL-6 and DNMT3 a and DNMT3 b protein in IPF group increased,compared with the corresponding indexes in the control group,P<0.05,the difference was statistically significant.The expressions of IL-1beta,IL-6,DNMT3 a and DNMT3 b were positively correlated with the expression of TGF-beta1 in IPF group.2.Compared with control group,the relative expression of TERT protein in peripheral blood leukocytes of experimental group increased,P<0.05,the difference was statistically significant.Conclusion:The levels of TGF-beta1,IL-1beta,IL-6 and DNMT3 a and DNMT3 b in peripheral blood of patients with IPF and the content of TERT in peripheral blood were higher than those in healthy controls.Part two: The effect of age factor on the expression of TERT、DNMT3a 、 DNMT3 b and TGF-beta1 in the model of pulmonary fibrosis in miceExperiment two: The effect of age factor on the expression of TERT and TGF-beta1 in the model of pulmonary fibrosis in miceObjective:To research the influence of age factor on the changes of TERT activity during IPF process and discusse the changes and significance of TGF-beta1,IL-1beta and IL-6 in IPF processMethods:128 10-12 week old male C57BL/6 mices,specific pathogen free SPF,were raised to 20 weeks of age,were randomly divided into young pulmonary fibrosis model group(group A),young control group(group B),pulmonary fibrosis model group(group C),aged control group(group D),each group 32 mices.The A group were injected with bleomycin(5mg/kg)into the pulmonary fibrosis model in the trachea of the mice.The B group was injected with the same dose of normal saline in the trachea for the control.The C and D groups were fed to the A group and the B group after 26 weeks of age to make the senile pulmonary fibrosis group model and the old normal control group.After the7 th,14th,21 th and 28 th days after the model,8 mices were randomly selected in group A,group B,group C and group D.After the blood extraction was executed with abdominal aorta,the specimens of lung tissue and blood samples were left to be measured.Lung tissue HE staining and Ashcroft score were used to evaluate the degree of pulmonary fibrosis;Masson staining was used to observe the distribution of collagen in lung tissue of mice;the Immune Histochemistry of E-Cad,Vimentin,alpha-SMA and gray value in mice lung tissue;and ELISA method to detect the content of TGF-beta1,IL-1 beta and IL-6 protein in mice plasma;Western Blot method was used to detect the TERT in lung tissue of mice.Results:1.BLM(5mg/kg)was injected into the trachea of mice,and the model of pulmonary fibrosis in mice was successfully established.Compared with young pulmonary fibrosis mice,The fibrosis degree in old pulmonary fibrosis group was statistically different from that in young mice,P<0.05.2.Compared with the control group,the expression of E-Cad in the model group decreased,alpha-SMA and Vimentin increased,and the difference was statistically significant.The three groups changed obviously with the progression of the disease.The differences between the three groups were 28 th days in the control group,14 th days in the model group and 28 th days in the model group.Compared with the young model group,the elderly model was compared with the old model mice.At the same time of modeling,the positive rate of E-Cad was low,and the positive rate of alpha-SMA and Vimentin was high in group A,P<0.05,the difference was statistically significant.3.The serum levels of TGF-beta1,IL-1beta and IL-6 in group A and group C were higher than that in group B and group D.The indexes of TGF-beta1,IL-1beta and IL-6were compared between the A group and the B group,the C group and the D group at the same time point,and the difference was statistically significant,P<0.05.but there was no statistical difference between group A and group C,group B and group D at the same time point,P>0.05.4.Compared with the corresponding time point B group and the D group,the relative expression of TERT protein in the lung tissue of the A group and the C group increased in 7th days,14 th days,21 th days and 28 th days,and the difference was statistically significant.Compared with the expression of seventh days,21 th days and28 th days respectively,the relative expression of TERT protein in the lung tissue of A group and C group increased,P<0.05,the difference was statistically significant.5.Compared with the corresponding time point B group and A group,the relative expression of TERT protein in lung tissue of D group and C group increased on the 14 th day and the 28 th day,P<0.05,the difference was statistically significant.The relative expression of TERT protein in lung tissue in group B was compared with that of twenty-eighth days in group B.The relative expression of TERT protein in lung tissue of group D was compared with that of twenty-eighth days in group D,and there was no statistical difference between them,P>0.05.6.In group A,the relative expression of TERT protein in the 14 th days of lung tissue,the relative expression of TERT protein in the lung tissue in the group A,and the relative expression of TERT protein in the lung tissue in the 14 th days or 28 th days in the group B were compared with any of the three,P<0.05,the difference was statistically significant.In group C,the relative expression of TERT protein in 14 th days of lung tissue,the relative expression of TERT protein in the lung tissue in the group C,and the relative expression of TERT protein in the lung tissue in 14 th days or 28 th days in the group D were compared,and the difference between the two groups was statistically significant in the three cases of the TERT protein expression in the lung tissue 14 th days or 28 th days.7.The relative expression level of TERT protein in lung tissue of group C and group A reached the peak on the fourteenth day,and decreased significantly on the twenty-eighth day.But compared with group A,the relative expression of TERT protein in group C fluctuated greatly.Conclusion:1.In the process of pulmonary fibrosis in mice,the E-Cad content of lung tissue decreased,the content of alpha-SMA and Vimentin increased,while the pulmonary fibrosis in the aged mice was more severe.The levels of TGF-beta1,IL-1beta and IL-6 in serum of pulmonary fibrosis mice increased significantly during the process of pulmonary fibrosis.TGF-beta1,IL-1beta and IL-6 may participate in the process of pulmonary fibrosis.2.The activity of TERT in lung tissue of pulmonary fibrosis mice increased first and then decreased.The change of TERT in the lung tissue of older pulmonary fibrosis mice is larger than that in young pulmonary fibrosis mice.Age factor may alter the degree of pulmonary fibrosis in mice by affecting the activity of TERT in lung tissue of mice with pulmonary fibrosis.Experiment three: The effect of age factor on the expression of DNMT3a and DNMT3 b in the model of pulmonary fibrosis in miceObjective:To research the influence of age factor on the expression of DNMT3 a,DNMT3b and CDH1 in IPF process.Methods:32 10-12 week old male C57BL/6 mices,specific pathogen free SPF,were raised to20 weeks of age,were randomly divided into young pulmonary fibrosis model group(group A),young control group(group B),pulmonary fibrosis model group(group C),aged control group(group D),each group 8,group A mice received intrateacheal injection of bleomycin pulmonary fibrosis model making dilution;were injected the same dose of saline group B trachea,group C and group D were raised to 26 weeks of age after respectively using group A and group B modeling method of manufacturing senile pulmonary fibrosis group model and elderly normal control group model.Anesthesia and modeling methods and Experiment two were similar in mice anesthesia and modeling methods.After group C and group D were raised to 26 weeks old,they were matched with group A and group B to create senile pulmonary fibrosis group model and elderly normal control group model respectively.The mice in group A,group B,group C and group D were killed by abdominal aorta blood sampling at twenty-eighth days after the model was made,and the specimens of lung tissue were left to be measured.HE staining and Ashcroft score in lung tissue of mice were evaluated the degree of pulmonary fibrosis;observation of collagen distribution in lung tissue of mice by Masson staining;Western Blot assay was used to detect the protein content of DNMT3 a,DNMT3b and CDH1 in lung tissue of mice.Results:1.Compared with group B,the expression of DNMT3 a and DNMT3 b protein in group A increased significantly,and the difference was statistically significant,P<0.05.Compared with group D,the expression of DNMT3 a and DNMT3 b protein in group C increased significantly,the difference was statistically significant,P<0.05.Compared with group A,the expression of DNMT3 a and DNMT3 b protein in group C increased significantly,and the difference was statistically significant,P<0.05.Compared with group B,there was no significant change in DNMT3 a and DNMT3 b protein expression in group D,the difference was not statistically significant,P>0.05.2.Compared with group B,the expression of CDH1 protein in group A decreased significantly,and the difference was statistically significant,P<0.05.Compared with group D,the expression of CDH1 protein in group C decreased significantly,the difference was statistically significant,P<0.05.Compared with group A,the expression of CDH1 protein in group C decreased significantly,and the difference was statistically significant,P<0.05.Compared with group B,there was no significant change in CDH1 protein expression in group D,the difference was not statistically significant,P>0.05.Conclusion:The expression of DNMT3 a and DNMT3 b increased and CDH1 decreased in pulmonary fibrosis mice.Age factor may affect the degree of pulmonary fibrosis by affecting the expression of DNMT3 a,DNMT3b and CDH1 in lung tissue of mice.Part three: The research on TGF-beta1 induced abnormal expression of TERT and DNMT3 a and DNMT3 b in A549 human alveolar type II epithelial cells and the intervention of DNMT3 a si RNA interference and TERT over-expressionExperiment four: The effect of TGF-beta1 on the expression of DNMT3 a,DNMT3b,CDH1,TERT and IL-1beta and IL-6 in A549 human alveolar type II epithelial cellsObjective:To research The effect of TGF-beta1 on the expression of DNMT3 a,DNMT3b,CDH1,TERT and IL-1beta and IL-6 in A549 human alveolar type II epithelial cells.Methods:The A549 human alveolar type II epithelial cells were cultured and divided into control group,TGF-beta1 2μmol/L intervention group,TGF-beta1 5μmol/L intervention group and TGF-beta1 10μmol/L intervention group.The effect of TGF-beta1 gradient stimulation on the senescence of A549 human alveolar type II epithelial cells by beta-gal staining;ELISA detection of TGF-beta1 Gradient stimulation on the expression of IL-1beta and IL-6 protein in A549 human alveolar type II epithelial cells;Quantitative Real-time PCR detection the expression of TGF-beta1 Gradient stimulation on the expression of IL-1 beta and IL-6 m RNA in A549 human alveolar type II epithelial cells;Observe the effect of TGF-beta1 on the morphology and collagen expression of A549 human alveolar type II epithelial cells;Quantitative Real-time PCR detection the expression of TGF-beta1 on COL-I,FN1,Tensin-C m RNA in A549 human alveolar type II epithelial cells;Quantitative Real-time PCR detection the expression of TGF-beta1 on DNMT3 a,DNMT3b,CDH1 and TERT m RNA in A549 human alveolar type II epithelial cells;Western Blot assay was used to detect the effect of TGF-beta1 on the expression of TERT protein in A549 human alveolar type II epithelial cells.Results:1.There was almost no aging cells in the normal control group,and the percentage of cell senescence in the intervention group increased with the increase of TGF-beta1 concentration.2.Compared with the control group,the expression of IL-1beta and IL6 protein and m RNA in A549 human alveolar type II epithelial cells increased significantly in the TGF-beta1 2μmol/L intervention group,TGF-beta1 5μmol/L intervention group and TGF-beta1 10μmol/L intervention group compared with the control group,the difference was statistically significant,P<0.05.With the increase of TGF-beta1 concentration,the expression of IL-1beta,IL6 protein and m RNA in A549 human alveolar type II epithelial cells also increased further.TGF-beta1 5μmol/L intervention group and TGF-beta110μmol/L intervention group compared with TGF-beta1 2μmol/L intervention group,the expression of A549 in human alveolar epithelial cells type IL-1 beta and IL6 protein and m RNA increased significantly,the difference was statistically significant,P<0.05.But compared with the TGF-beta1 5μmol/L intervention group and TGF-beta1 10μmol/L intervention group,the difference was not statistically significant,P>0.05.3.Under the light microscope control group cells showed typical epithelial cell like morphology,TGF-beta1 10μmol/L intervention group cells showed typical interstitial cells in morphology,Masson staining was observed in the control group were almost no blue collagen expression,and TGF-beta1 10μmol/L intervention group cells appeared large blue collagen,and expression the expression range increased significantly.4.Compared with the control group,the expression of COL-I,FN1 and Tensin-C m RNA in A549 human alveolar type II epithelial cells increased significantly in the TGF-beta1 10μmol/L intervention group,the difference was statistically significant,P<0.05.5.Compared with the control group,the expression of DNMT3 a and DNMT3 b m RNA in A549 human alveolar type II epithelial cells increased significantly,and the expression of TERT and CHD1 m RNA decreased significantly in the TGF-beta110μmol/L intervention group compared with the control group,the difference was statistically significant,P<0.05.Compared with the control group,the expression of TERT protein in the human alveolar type II epithelial cells of TGF-beta1 10μmol/L intervention group was significantly lower than that in the control group,and the difference was statistically significant,P<0.05.Conclusion:1.TGF-beta1 can lead to aging of A549 human alveolar type II epithelial cells and promote the expression of IL-1beta,IL6,COL-I,FN1 and Tensin-C in A549 human alveolar type II epithelial cells.TGF-beta1 promotes the expression of DNMT3 a and DNMT3 b in A549 human alveolar type II epithelial cells and inhibits the expression of TERT and CHD1.2.TGF-beta1 promoted the expression of DNMT3 a and DNMT3 b in A549 human alveolar type II epithelial cells,and inhibited the expression of TERT and CHD1.Experiment five: The effect of TGF-beta1 on the activity of TERT promoter in A549 human alveolar type II epithelial cells and the effect of DNMT3 a,DNMT3b transcription factors and TERT promoterObjective:To research the effect of TGF-beta1 on the activity of TERT promoter in A549 human alveolar type II epithelial cells and the effect of DNMT3 a,DNMT3b transcription factors and TERT promoter.Methods:The A549 human alveolar type II epithelial cells were cultured and divided into the control group and the TGF-beta1 10μmol/L intervention group.Luciferase assay was used to detect the effect of TGF-beta1 on the activity of TERT promoter in A549 human alveolar type II epithelial cells.Ch IP(chromatin immunoprecipitation)was used to detect the effect of TGF-beta1 on the binding of DNMT3 a and DNMT3 b to TERT promoter in A549 human alveolar type II epithelial cells.Results:1.Compared with the control group,the activity of TERT promoter in A549 human alveolar type II epithelial cells decreased in the TGF-beta1 10μmol/L intervention group,and the difference was statistically significant,P<0.05.2.Compared with the control group,the relative binding of DNMT3 a and DNMT3 b transcription factors to TERT promoter in A549 alveolar type II epithelial cells increased in the TGF-beta1 10μmol/L intervention group,the difference was statistically significant,P<0.05.Compared the enrichment of DNMT3 a and DNMT3 b transcription factors.The enrichment of DNMT3 a transcription factors was much more than that of DNMT3 b transcription factors,and the difference was statistically significant,P<0.05.Conclusion:TGF-beta1 promotes the binding of DNMT3 a,DNMT3b transcription factors to TERT promoter,and DNMT3 a transcription factor plays a major role in the binding of TERT promoter.The methylation level of TERT promoter was affected,and the activity of TERT promoter decreased.Experiment six: Intervention of DNMT3 a si RNA interference and over-expression of TERT on TGF-beta1 induced fibrosis and senescence of A549 human alveolar type II epithelial cellsObjective:The intervention of DNMT3 a si RNA interference and TERT over-expression on the fibrosis and senescence of A549 human alveolar type II epithelial cells induced by TGF-beta1.Methods:Cultured A549 human alveolar type II epithelial cells were divided into control group,TGF-beta1 intervention group,TGF-beta1 intervention +DNMT3a si RNA interference group,TGF-beta1 intervention +TERT over-expression group and TGF-beta1 intervention +DNMT3a si RNA interference over expression group.si RNA interference on DNMT3 a,lentivirus as carrier,plasmid lentivirus transfection and over-expression of TERT were carried out.TGF-beta1 intervention +TERT over-expression group and TGF-beta1 intervention +DNMT3a si RNA interference+TERT over expression group was plasmid lentivirus transfection;control group,TGF-beta1 intervention group and TGF-beta1 intervention +DNMT3a si RNA interference group,LV-GFP plasmid lentivirus empty load control.Confocal laser scanning fluorescence microscopy was used to detect the fluorescence of A549 human alveolar type II epithelial cells in each group.Masson staining was used to detect the expression of collagen in A549 human alveolar type II epithelial cells.The effect of the expression of COL-I,FN1,Tensin-C m RNA in A549 human alveolar type II epithelial cells in each group by Quantitative R-T PCR.The influence of the expression of TERT,DNMT3 a,DNMT3b and CDH1 protein in A549 human alveolar type II epithelial cells in each group by Western Blot.The senescence of A549 human alveolar type II epithelial cells was detected by beta-gal staining.Results:1.DNMT3 a si RNA interference and TERT over-expression were succeed.2.After Masson staining,the cells in the control group almost no blue collagen expression,and TGF-beta1 intervention group cells appeared large blue collagen,its expression and expression range were significantly increased,compared with the control group,the difference was statistically significant,P<0.05.TGF-beta1 intervention+DNMT3a si RNA interference group,TGF-beta1 intervention of +TERT over-expression group and TGF-beta1 intervention +DNMT3a si RNA interference+TERT over-expression group were stained collagen expression,the three group was no difference,but compared with the control group,the blue collagen expression and expression range increased,the difference was statistically significant,P<0.05;the three groups respectively with TGF-beta1 intervention group compared to collagen expression and expression range were decreased,the difference was statistically significant,P<0.05.3.Compared with the control group,the expression of COL-I,FN1,Tensin-C m RNA in A549 human alveolar epithelial cells in TGF-beta1 intervention group was significantly higher than that in the control group,and the difference was statistically significant,P<0.05.The expression of COL-I,FN1,Tensin-C m RNA in A549 human alveolar epithelial cells in TGF-beta1 intervention +DNMT3a si RNA interference group,TGF-beta1 intervention of +TERT over-expression group and TGF-beta1 intervention+DNMT3a si RNA interference +TERT over-expression group were expression increased,the three group was no difference,but the difference compared with the control group,the COL-I,FN1,Tensin-C m RNA expression in three groups were increased,the difference was statistically significant,P<0.05;compared with the TGF-beta1 intervention group,the COL-I,FN1,Tensin-C m RNA expression in three groups were decreased,the difference was statistically significant,P<0.05.4.Compared with the control group,the expression of DNMT3 a and DNMT3 b protein in A549 human alveolar type II epithelial cells increased significantly,and the expression of TERT and CDH1 protein decreased significantly in the TGF-beta1 intervention group,and the difference was statistically significant,P<0.05.The expression of DNMT3 a and DNMT3 b protein increased and TERT and CDH1 protein decreased in A549 human alveolar epithelial cells in TGF-beta1 intervention +DNMT3a si RNA interference group,TGF-beta1 intervention of +TERT over-expression group and TGF-beta1 intervention +DNMT3a si RNA interference +TERT over-expression group,the three group was no difference,but the difference compared with the control group,the DNMT3 a and DNMT3 b protein increased and TERT and CDH1 protein decreased in three groups,the difference was statistically significant,P<0.05;compared with the TGF-beta1 intervention group,the DNMT3 a and DNMT3 b protein decreased and TERT and CDH1 protein increased in three groups,the difference was statistically significant,P<0.05.5.There were almost no senescent cells in the control group,while a large number of senescent cells appeared in the TGF-beta1 intervention group,and the proportion of senescent cells was the largest.In TGF-beta1 intervention +DNMT3a si RNA interference group,TGF-beta1 intervention of +TERT over-expression group and TGF-beta1 intervention +DNMT3a si RNA interference +TERT over-expression group,the three groups were expressed the aging cells,there was no difference in the proportion of aging cells between the three groups,However,compared with the control group,the three groups increased and increased the number of aging cells.The difference between the three groups was statistically significant.Compared with the TGF-beta1 intervention group,the aging cells decreased and the proportion decreased,the difference was statistically significant,P<0.05.Conclusion:Interfering with DNMT3 a si RNA and overexpression of TERT could reduce the fibrosis effect of TGF-beta 1 on A549 human alveolar type II epithelial cells and the degree of aging of A549 human alveolar type II epithelial cells.However,there was no overlap between DNMT3 a si RNA interference and TERT overexpression. |
PDF Full Text Request