Effects Of Demethylation On The Emphysema Animal Model, Endothelial Progenitor Cells’ Function And Stem Cell Antigen-1’s Expression

Chapter1Effect of AZA on CSE induced emphysema animal model and Sca-l’s expressionObjectiveTo establish and evaluate emphysema murine model induced by intraperitoneal injection of CSE. To investigate the protective effect of AZA on emphysema murine model and Sca-1’s expression.MethodsFour to six weeks old male C57BL/6J mice were divided into four groups:controls, AZA group, CSE+AZA group and CSE group(n=8each group). The total experimental period was four weeks with timing and quantitive intraperitoneal injection of CSE, AZA and PBS respectively. At day28, the mice were disposed for body weighting, lung function measured by PLY3211system, pathomorphological changes of lung tissue by HE staining, and separation of bone marrow-derived EPCs by percoll density gradient centrifugation. MAST, MLI, DI and the apoptosis of alveolar interval cells were calculated. Sca-1protein expression in bone marrow-derived EPCs and lung tissue were detected by Western-blotting. Sca-1mRNA expression in bone marrow-derived EPCs and lung tissue were detected by real-time RT-PCR.Results (1)(ⅰ) There was no statistical difference among4groups in weight (P>0.05). The Raw of AC group and C group were higher than that of controls(P<0.01). The Cdyn, PEF, Ti/Te of AC group and C group were lower than those of controls (P<0.05or P<0.01). There were no difference between AC group and C group in Raw, Cdyn, PEF and Ti/Te (P>0.05).(ⅱ) Morphological detection of lung tissues showed significant disruption and thinning of alveolar septum, enlarged airspace and formation of emphysema in C group. Quantitative morphological analysis showed:MAST of A, AC and C group were lower than that of controls(P<0.01). MAST of AC and C group were lower than that of A group(P<0.01). MAST of C group was lower than that of AC group(P<0.05). MLI and DI of A, AC, C groups were higher than those of controls(P<0.05or P<0.01). MLI and DI of AC, C groups were higher than those of A group(P<0.01). MLI and DI of C group were higher than those of AC group(P<0.05).(ⅲ) AI of AC group and C group were higher than that of controls or A group(P<0.01). AI of C group was higher than that of AC group(P<0.01).(2)(i)Sca-1protein expression in EPCs of AC group and C group were lower than that of controls or A group(P<0.05or P<0.01). Sca-1protein expression in C group was lower than that of AC group(P<0.01).Sca-1mRNA expression in EPCs of AC and C groups were lower than that of controls or A group(P<0.01). Sca-1mRNA expression in EPCs C groups was lower than that of AC group(P<0.05)(ii) Sca-1protein expression in lung tissue of A, AC and C group were lower than that of controls(P<0.01). Sca-1protein expression in lung tissue of AC and C group were lower than that A group(P<0.01). Sca-1protein expression in lung tissue of C group was lower than that of AC group(P<0.01). Sca-1mRNA expression in lung tissue of A, AC and C group were lower than that of controls(P<0.01). Sca-1protein expression in lung tissue AC and C group were lower than that of A group(P<0.01). Sca-1protein expression in lung tissue C group was lower than that of AC group(P<0.05)Conclusions(1) Intraperitoneal injection of CSE can establish emphysema murine model and shorten the period of modelling.. The model is similar to human COPD. The morphological changes of lung tissue occur earlier than the change of lung function.(2) DNA methyltransferase inhibitor may protect lung function and histomorphology by partly reverse Sca-1suppression induced by CSE.(3) Epigenetics DNA methylation may be involved in the emphysema forming process induced by CSE. Chapter2Influences of5-aza-2’-deoxycytidine (AZA) on function and stem cell antigen-1(Sca-1) on endothelial progenitor cells(EPCs) interfered by cigarette smoke extract(CSE)ObjectiveTo observe the stem cell antigen-1(Sca-1) gene expression on murine endothelial progenitor cells(EPCs).To investigate the influences of5-aza-2’-deoxycytidine (AZA) on function and stem cell antigen-1(Sca-1) on endothelial progenitor cells(EPCs) interfered by cigarette smoke extract(CSE). MethodsMononuclear cells were isolated from murine bone marrow by Ficoll density gradient centrifugation and cultured with endothelial growth medium-2(EGM-2). EPCs were identified by morphology, dual staining for Dil-acLDL and FITC-UEA-1, surface markers analysed by flow cytometry including CD34, CD133and Flk-1on the7th day of culture. The expression of Sca-1on EPCs was also analysed by flow cytometry.Selected the suitable intervention concentration and timing from various intervention concentration and timing of CSE and AZA in vitro, and then used them to proceed with subsequent experiments. The proliferation capacity of EPCs was detected by MTT assay, the adhesive capacity was assessed by adherent cell count, and the secretion capacity was measured by the concentration of NO in EPCs culture fluid.EPCs were divided into four groups:control group(N), AZAgroup(A), AZA plus CSE group(AC) and CSE group(C), each group took the respective intervention. The Sca-1protein was detected by Western-blotting.Results(1)Cells on the7th day of culture showed fusiform, polygon, forming capillary structure or "cobblestone" morphology. The Dil-acLDL and FITC-UEA-1amphophilic cells’positive rate was (94.67±4.16)%. The concurrent positive expression rate of FITC-CD34、PE-CD133and APC-Flk-1was (95.07±1.73)%. The concurrent positive expression rate of FITC-CD34、PE-CD133、APC-Flk-1and PerCP-Sca-1was (94.00±1.67)%. The positive expression rate of Sca-1in EPCs was (98.87±0.24)%. (2)(i) When CSE interfered EPCs for3hours, the OD values of1%CSE group and2.5%CSE group were higher than that of control group(P<0.05), and the OD values of5%CSE group and10%CSE group were lower than that of control group (P<0.01). When CSE interfered EPCs for6hours, the OD value of1%CSE group was higher than that of control group(P<0.05), and the OD values of2.5%CSE group,5%CSE group and10%CSE group were lower than that of control group (P<0.01). When CSE interfered EPCs for24hours, the OD values in all the CSE groups were lower than that of control group(P<0.01).(ⅱ) The OD values of1%CSE group,1%CSE+2umol/L AZA group,1%CSE+5umol/L AZA group were lower than that of control group(P<0.01). The OD values of1%CSE+2umol/L AZA group,1%CSE+5umol/L AZA group were higher than that of1%CSE group(P<0.01). There was no statistical difference between1%CSE+2umol/L AZA group and1%CSE+5umol/L AZA group in the OD value (P>0.05).(ⅲ) The adherent cells counts of1%CSE group and1%CSE+2umol/L AZA group were lower than that of control group(P<0.01). The adherent cells count of1%CSE group was lower than that of1%CSE+2umol/L AZA group(P<0.01). The concentrations of NO of1%CSE group and1%CSE+2umol/L AZA group were lower than that of control group(P<0.01). There was no statistical difference between1%CSE+2umol/L AZA group and1%CSE+5umol/L AZA group in the concentration of NO (P>0.05)(3) Compared with the control group, Sca-1protein expression decreased significantly in CSE group (P<0.01). There was no statistical difference among control group, AZA group and CSE+AZA group in Sca-1protein expression (P>0.05). Compared with amongCSE group, Sca-1protein expression increased significantly in AZA group and CSE+AZA group(P<0.01). There was no statistical difference between AZA group and CSE+AZA group in Sca-1protein expression (P>0.05)Conclusions(1) There is a high expression rate of Sca-1in murine EPCs.(2) EPCs’proliferation capacity increases compensatorily with the low concentration and short-term CSE intervention. But with the increasing concentration and intervening time of CSE, EPCs’capacity including proliferation, adhesion and secretion will decrease and even be suppressed absolutely.(3) DNA methyltransferase inhibitor can partly reverse Sca-1protein suppression in EPCs induced by CSE, and at the same time improves EPCs’ functions. Sca-1protein expression has the same tendence with EPCs’ capacities intervened by CSE. DNA methylation may be involved in the dysfunction of EPCs induced by CSE. Sca-1DNA methylation may contribute to the dysfunction of EPCs induced by CSE.