| Part 1:Changes of EPCs function in peripheral blood of COPD ratsObjectiveRats model of chronic obstructive pulmonary disease were replicate?Rat peripheral blood endothelial progenitor cells were extracted and identified in vitro.The effects of COPD on proliferation,adhesion,migration,cycle and apoptosis of endothelial progenitor cells were observed.MethodsFirstly,replicate rat model of COPD with smudging plus LPS,and observe the changes of lung structures by counting the inflammatory cells in lung lavage fluid and HE staining to judge whether the replication of COPD rats are successful.After collection of blood from the abdominal aorta,mononuclear cells were obtained from peripheral blood of rats by density gradient centrifugation and inoculated on six-well plates with cell slides.Cells were cultivated using EBM-2special culture medium until cell attachment.After adherent differentiation,the cells were identified by used labeled with adsorption of UEA-1and endocytosis of Dil-ac-LDL by FITC.The CCK-8 kit was used to identify the proliferation and adhesion functions of EPCs.The cell migration function was observed by transwell chamber,and the cycle and apoptosis of EPCs were detected by flow cytometry.ResultsThrough the smoke-injection combined with LPS method,inflammatory cell counts and pathological findings in bronchoalveolar lavage fluid proved that the COPD rat model successfully replicated.Identification of isolated cultured cells as EPCs.The proliferative capacity of EPCs in the COPD rat group was significantly lower than that in the control group by the CCK-8 method?P<0.01?.Adhesion ability of EPCs in group COPD was significantly lower than that in control group?P<0.01?.The migration ability of EPCs in group COPD was significantly lower than that in control group by transwell?P<000?.Compared with the control group,COPD group significantly increased G0G1 EPCs?P<0.01?,and significantly decreased EPCs at S and G2M stages?P<0.01;P<0.05?.COPD group apoptosis rate was significantly higher than that of the control group?P<0.05?.Data statistics were analyzed with SPSS20 statistical software.The data was expressed as ???±s.T-test was used for comparison between groups.P<0.05 represented statistically significant difference.ConclusionsThe proliferation,migration and adhesion of EPCs in peripheral blood of COPD rats were decreased.The cell cycle arrest was decreased in G0G1phase,S phase and G2M phase,and the apoptosis rate was significantly increased.Part 2:Effect of Sirt1 on Propagation,Migration,Adhesion,Cycle and Apoptosis of EPCsChapter 1:EPCs Express Sirt1Objective It is clear that Sirt1 is expressed in EPCs.MethodsPeripheral blood EPCs were cultured in vitro in COPD rats and control groups.EPCs expressed Sirt1 by PCR,Western blot and immunofluorescence.Data statistics were analyzed with SPSS20 statistical software.The data was expressed as ???±s.T-test was used for comparison between groups.P<0.05 represented statistically significant difference.ResultsThe expression of Sirt1 mRNA in EPCs of COPD rats was significantly lower than that in control cells?P<0.01?.The Sirt1 protein band was detected in both COPD and control EPCs.The expression of Sirt1 protein in the COPD rat normalized by?-actin was significantly lower than that in the control group by WCIF ImageJ image analysis software?P<0.01?.In light of observation of confocal microscope of Sirt1 in COPD rats and control group,Sirt1 was expressed in both groups,and the fluorescence intensity of Sirt1 in COPD rats was significantly lower than that in control group.ConclusionsSirt1 was expressed in EPCs of COPD group and control group,and COPD group was significantly lower than that of control group.Chapter 2:Effect of Sirt1 on Propagation,Migration,Adhesion,Cycle and Apoptosis of EPCsObjectiveObserve the effect of Sirt1 on proliferation,adhesion,migration,cycle and apoptosis of EPCs.MethodsThe use of Sirt1 agonist?SRT1720?and transfection of EPCs carrying Sirt1 virus lentivirus resulted in overexpression of Sirt1 as an agonist group and the use of Sirt1 inhibitor?EX527?to reduce Sirt1 expression in EPCs as an inhibitor group.The CCK-8 kit was used to identify the proliferation and adhesion functions of EPCs.The cell migration function was observed by transwell chamber,and the cycle and apoptosis of EPCs were detected by flow cytometry.ResultsAgonist and Sirt1 lentivirus transfection significantly increased the proliferation of EPCs after Sirt1 expression compared with COPD group?P<0.05;P<0.05?.The proliferation ability of inhibitor group was significantly lower than that of COPD group?P<0.05?.The adhesion ability of EPCs in agonist group was significantly higher than that in COPD?P<0.01?.The adhesion ability of inhibitor group was lower than that of COPD group,but there was no statistical significance?P>0.05?.The mobility of EPCs in the agonist group was significantly lower than that in COPD?P<0.0001?,and the migration ability in the inhibitor group was significantly lower than that in the COPD group?P<0.001?.Compared with the COPD group,the G0G1 EPCs in the agonist group were significantly decreased?P<0.001?,and the EPCs in the S and G2M stages were significantly increased?P<0.01;P<0.01?.The inhibitor group was significantly more than the COPD group in the G0G1 EPCs?P<0.05?.),EPCs in S and G2M decreased,but there was no statistical significance?P>0.05;P>0.05?.The apoptosis rate of EPCs in agonist group was significantly lower than that in COPD group?P<0.05?.The apoptosis rate of EPCs in inhibitor group was significantly higher than that in COPD group?P<0.01?.Data statistics were analyzed with SPSS20 statistical software.The data was expressed as ???±s.T-test was used for comparison between groups.P<0.05represented statistically significant difference.ConclusionsOverexpression of Sirt1 promotes the proliferation,adhesion and migration of EPCs and reduces apoptosis.When Sirt1 expression is decreased,the proliferation,adhesion,and migration of EPCs are inhibited and the apoptosis is increased.Part 3:Effect of Sirt1 on NF-KB,FOXO3a,P53 GenesObjectiveThe expression of Sirt1,FOXO3a,p53,and NF-kB in EPCs of COPD rats were observed after changing Sirt1 expression.A rat model of chronic obstructive pulmonary disease was replicated and the relationship between the expression of SIRT1 with FOXO3a,p53,and NF-kB was observed.The relationship between Sirt1and FOXO3a,NF-KB and P53 was investigated.MethodsThe expressions of Sirt1,FOXO3a,NF-KB and P53 were detected;Cell slides were prepared,and the expression of each protein in EPCs was observed by immunofluorescence.After removing the lung tissue from successfully replicated COPD rats,mRNA and protein were extracted and paraffin-embedded;the expression of Sirt1,FOXO3a,NF-KB,and P53 in lung tissue was detected by qPCR,Western blot,and immunofluorescence.Data statistics were analyzed with SPSS20 statistical software.The data was expressed as ???±s.T-test was used for comparison between groups.P<0.05 represented statistically significant difference.ResultsThe second part:The relative mRNA expression of Sirt1 in EPCs in COPD group was significantly lower than that in the control group by qPCR?P<0.01?;The expression of Sirt1 mRNA in agonist group was significantly higher than that in COPD group?P<0.01?;The expression of Sirt1 mRNA in inhibitor group was significantly lower than that in COPD group?P<0.05?.The relative mRNA expression of FOXO3a in EPCs was significantly lower in the COPD group than in the control group by qPCR?P<0.01?.The expression of FOXO3a mRNA in agonist group was significantly higher than that in COPD group?P<0.01?.The expression of FOXO3a mRNA in inhibitor group was significantly lower than that in COPD group?P<0.05?.The relative expression of NF-KB mRNA in EPCs in the COPD group was significantly higher than that in the control group by qPCR?P<0.01?.The expression of NF-KB mRNA in agonist group was significantly lower than that in COPD group?P<0.05?.The expression of NF-KB mRNA in inhibitor group was significantly lower than that in COPD group?P<0.0001?.The relative mRNA expression of EPCs P53 in the COPD group was significantly higher than that in the control group by qPCR?P<0.001?.The expression of P53 mRNA in agonist group was significantly lower than that in COPD group?P<0.001?.The expression of P53 mRNA in inhibitor group was significantly lower than that in COPD group?P<0.01?.Western blote showed that the expression of Sirt1 protein in EPCs in COPD group was significantly lower than that in the control group?P<0.01?.The expression of Sirt1 protein in agonist group was significantly higher than that in COPD group?P<0.01?.The expression of Sirt1protein in inhibitor group was significantly lower than that in COPD group?P<0.001?.Western blote showed that the expression of FOXO3a protein in EPCs in COPD group was significantly lower than that in the control group?P<0.001?.The expression of FOXO3a protein in agonist group was significantly higher than that in COPD group?P<0.001?.The expression of FOXO3a protein in inhibitor group was significantly lower than that in COPD group?P<0.001?.Western blote showed that the expression of NF-KB protein in EPCs in COPD group was significantly higher than that in control group?P<0.01?.The expression of NF-KB protein in agonist group was lower than that in COPD group,but there was no statistical significance?P>0.05?.The expression of NF-KB protein in inhibitor group was significantly higher than that in COPD group?P<0.05?.Western blote showed that the expression of P53protein in EPCs in COPD group was significantly higher than that in control group?P<0.001?.The expression of P53 protein in agonist group was significantly lower than that in COPD group?P<0.001?.The expression of P53 protein in inhibitor group was significantly higher than that in COPD group?P<0.01?.Cellular immunofluorescence showed that the expression of Sirt1 in COPD group was significantly lower than that in the control group.The expression of Sirt1in agonist group was significantly higher than that in COPD group.The expression of Sirt1 in EPCs in inhibitor group was significantly lower than that in COPD group.Cellular immunofluorescence showed that the expression of FOXO3a in EPCs in COPD group was significantly lower than that in the control group.The expression of FOXO3a in EPCs in agonist group was significantly higher than that in COPD group.The expression of Sirt1 in EPCs in inhibitor group was significantly lower than that in COPD group.Cellular immunofluorescence showed that expression of NF-KB in EPCs was significantly increased in the COPD group compared with the control group.The expression of NF-KB in EPCs in agonist group was significantly lower than that in COPD group.The expression of NF-KB in EPCs in inhibitor group was significantly higher than that in COPD group.Cellular immunofluorescence showed that the expression of P53 in EPCs was significantly increased in COPD group compared with the control group.The expression of P53 in EPCs in agonist group was significantly lower than that in COPD group.The expression of P53 in EPCs in inhibitor group was significantly higher than that in COPD group.At the same time,we found that the relative expression of Sirt1 mRNA in the COPD group was significantly lower than that in the control group?P<0.05?,and the mRNA expression of FOXO3a in the COPD group was also significantly lower than that in the control group?P<0.05?.The expression of Sirt1 mRNA in lung tissue was significantly higher in KB than in the control group?P<0.001?.The expression of P53mRNA in lung tissue was also significantly higher than that in the control group.The expression of SIRT1 protein in lung tissue of COPD group was significantly lower than that of control group?P<0.05?.The expression level of FOXO3a in lung tissue was also significantly lower than that of control group?P<0.05?;and the expression of NF-KB protein in lung tissue.The COPD group was significantly higher than the control group?P<0.0001?;P53 expression in the lung tissue of the COPD group was significantly reduced?P<0.05?.The expression of SIRT1 in lung tissue of COPD group was significantly lower than that of control group by immunofluorescence method?P<0.001?;the expression of FOXO3a in lung tissue of COPD group was significantly lower than that of control group?P<0.001?,and the expression of NF-KB in COPD group.The amount of P53 was significantly higher than that of the control group?P<0.001?.The expression of P53 in the COPD group was also significantly higher than that in the control group?P<0.05?.ConclusionsThe effect of Sirt1 on the function of EPCs is mainly through the Sirt1/FOXO3a/NF-KB/P53 signaling pathway,but P53 protein may also be regulated by other proteins in COPD rats. |
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