Exosome Derived From GATA4 Overexpressing Mouse Bone Marrow Mesenchymal Stem Cells Improve Myocardial Infarction Function Effection And Mechanism

Part Ⅰ: Exosome derived from GATA4 overexpressing mouse bone marrow mesenchymal stem cells improve myocardial infarction function effectionObjective: To determine that the ability of mouse bone marrow mesenchymal stem cells of overexpressing GATA4 secrete exosome differentiation into cardiomyocytelike cells and anti apoptosis in vitro.And to further validate the results in vitro.Methods: 1、In vitro by GATA4-BMSCs secrete exosome was co-cultured with BMSCs,empty vector-BMSCs secrete exosome was co-cultured with BMSCs,BMSCs secrete exosome was co-cultured with BMSCs,BMSCs was cultured alone and mice myocardial cells was cultured alone for 24 hours,48 hours,72 hours,and then the first qualitative immunofluorescent detection of myocardial specific antigen c Tn T,alpha-actin,connexin 43 and Desmin.And the method of RT-PCR was used to detect the expression of cardiac specific antigen c Tn T,-actin,connexin 43 and Desmin.2、Serum free culture GATA4 BMSCs secrete exosome co-cultured with myocardial cells,empty vector-BMSCs secrete exosome co-cultured with myocardial cells,BMSCs secrete exosome co-cultured with myocardial cells and myocardial cells cultured alone at hypoxia(1%)culture induced apoptosis.At the same time normal conditions to cultivate myocardial cells as a control group.The above groups were cultured for 24,48,72 hours to induce apoptosis.Flow cytometry was used to detect the apoptosis rate of cells in each group and the Westernblot technique was used to detect Caspase-3,Caspase-9,beta-actin and cytochrome C in each time period.3、 After 48 hours of mouse myocardial infarction model,then intra tail venous injection of Dir pre stained GATA4-BMSCs secrete exosome,empty vector-BMSCs secrete exosome,BMSCs secrete exosome.Mouse myocardial infarction model and normal feeding will not be given any treatment as the control group.The cardiac function was improved in 48 h,72h and 96h after intervention by color Doppler examination.Animal vivo imaging was used to detect the fluorescence intensity in myocardial infarction.Tomato Lectin was evaluated for local angiogenesis of myocardial infarction.By tunnel Assessment of apoptosis and the number of C-kit positive cells was assessed by immunohistochemistry.Results: 1、The expression of cTnT,α-actin,connexin-43 and desmin in GATA4-BMSC secrete exosome co-cultured with BMSCs were much higher than those in other groups,and with the time prolonging its expression increased.It is proved that GATA4 secrete exosome can effectively promote BMSCs differentiation into cardiomyocytelike cells.2、The results of flow cytometry showed that the GATA4-BMSCs secrete exosome group had stronger anti-apoptotic ability,but the apoptosis rate increased with time,but it was lower than other groups.Westernblot results showed that: In hypoxia no serum culture 24 h,48h,72 h GATA4-BMSCs secrete exosome group Caspase8,Cytochrome C expression was the lowest than other groups.But there was no significant difference with the normal culture group.But there was no significant difference in the expression of Caspase3,Caspase 9 among the groups.It is suggested that GATA4 may play an important role in the Cytochrome C pathway and Fas pathway.In vivo experiment can be seen: GATA4-BMSCs secrete exosome group heart function improved more significant than other groups.Ejection fraction and Ring contracted fraction is significantly improved before and after GATA4-BMSCs secrete exosome treatment myocardial infarction,untreated group cardiac function has deteriorated.Then animal vivo imaging to detect in 48 hours,72 hours and 96 hours the myocardial infarction,GATA4-BMSCs secrete exosome group than other groups,local Dir fluorescence intensity.Myocardial infarction control group and normal heart local fluorescence intensity is zero.Tomato Lectin experiment shows: GATA4-BMSCs secrete exosome group can express more blood vessels than other groups in the intervention after 48 hours,72 hours,96 hours.With the prolong of time,the number of blood vessels increased,but the increase is not significant.Tunnel experiment: GATA4-BMSCs secrete exosome group the number of apoptotic cells in the myocardial infarction group was less than other groups in the intervention measures after 48 hours,72 hours,96 hours.With the prolong time,the number of apoptotic cells increased,but the increase was not significant.C-kit immunohistochemical showed that GATA4-BMSCs secrete exosome the number of cardiac stem local C-kit cells more than other groups in the intervention after 48 hours,72 hours,96 hours,With the prolong of time,the number of C-kit cells increased,but the increase was not significant.Conclusion: Overexpressing GATA4 BMSCs secrete exosome can promote the BMSCs differentiation into cardiomyocyte-like cells,enhance the local aggregation of BMSCs in myocardial infarction,promote the local vascular development of myocardial infarction,and effectively mobilize C-kit positive cells,at multiple levels improve myocardial function after myocardial infarction.Part Ⅱ: Using Agilent micro RNA chip detect GATA4 overexpressing mouse bone marrow mesenchymal stem cells secreting exosome include micro RNAs related to cell differentiation and anti-apoptoticObjective: To detect cell differentiation and anti apoptosis micro RNA in exosome that secreted by overexpressing GATA4 mouse bone marrow mesenchymal stem cells.And using Agilent micro RNA chip to complete it.Methods: Obtain three Agilent micro RNA chips which about overexpressing GATA4 BMSCs secrete exosome,empty vector-BMSCs secrete exosome,BMSCs secrete exosome.Obtain original data.Using GO,Pathway and other bioinformatics processing data,The micro RNAs involved in cell differentiation and apoptosis were extracted.Results: The screening criteria for p≤5%,At the same time FC(ABS)more than 2 times is considered as significant difference,And at the same time need to meet: There was no significant difference in the expression of exosome that secreted by empty vector and BMSCs.However,GATA4-BMSCs secrete exosome and exosome secreted by BMSCs had the same trend as GATA4-BMSCs secrete exosome and exosome secreted by empty vector BMSCs,but it expression different trend of micro RNA empty vector BMSCs secrete exosome and BMSCs secrete exosome.There are 102 such micro RNAs.Which involved micro RNAs associated with cell differentiation,8 upregulated and 5 downregulated.Involving anti apoptotic micro RNA,a total of 21 micro RNA are up-regulated,the 4 micro RNA are down regulated.To further complete the validation of micro RNA,we extend the difference between the above screening criteria to 20 and the above conditions still need to meet.A total of 5 micro RNA were up-regulated in this micro RNA,and the 6 micro RNA was downregulated.Then the above predicted micro RNA was associated with differentiation,anti apoptosis,migration related,and the have significant difference expression micro RNA make a intersection.The key micro RNAs involved in cell differentiation are: mmu-mi R-199a-3p,This micro RNA is up-regulated;mmu-mi R-1894-5p,this micro RNA is downregulated.The key micro RNAs involved in cell apoptosis are: mmu-mi R-199a-3p,mmu-mi R-20a-5p and mmu-mi R-330-3p,all of which are up-regulated.We can conclude that mmu-mi R-199a-3p is involved in cell differentiation and involves cell anti-apoptotic,which is the key validation of micro RNA.Conclusion: Using Agilent microRNA chip detection mouse bone marrow mesenchymal stem cells overexpressing GATA4 secrete exosome that about cell differentiation and anti apoptosis micro RNA.Final confirmation of the first need to focus on verification of micro RNA is mmu-mi R-199a-3p.Part Ⅲ: Using TMT labeled quantitative proteomic analysis mouse bone marrow mesenchymal stem cells overexpressing GATA4 secrete exosome that about cell differentiation and anti apoptosis proteinsObjective: To detect cell differentiation and anti apoptosis proteins in exosome that secreted by overexpressing GATA4 mouse bone marrow mesenchymal stem cells.And using Tandem mass tag(TMT)labeled quantitative proteomic analysis to complete it.Methods: Using TMT labeled protein in exosome that secreted by overexpressing GATA4 BMSCs,empty vector BMSCs,BMSCs.Obtain original data.Using GO,Pathway and other bioinformatics processing data.Extraction of proteins involved in cell differentiation and apoptosis.Results: A total of 1539 proteins were identified in this study,of which the 1285 proteins contained quantitative information.Assuming ratio>1.2,the protein is upregulated,Ratio<0.83,that the protein down regulated,GATA4-BMSCs secrete exosome compared with BMSCs secrete exosome,among 77 proteins were upregulated,61 proteins were down-regulated.GATA4-BMSCs secrete exosome compared with empty vector-BMSCs secrete exosome,among 55 proteins were upregulated,164 proteins were down-regulated.Empty vector-BMSCs secrete exosome compared with BMSCs secrete exosome,among 116 proteins were up-regulated,92 proteins were down-regulated.The following screening criteria are used: There was no significant difference in the expression proteins of exosome that secreted by empty vector and BMSCs.However,GATA4-BMSCs secrete exosome and exosome secreted by BMSCs had the same trend as GATA4-BMSCs secrete exosome and exosome secreted by empty vector BMSCs,but it expression different trend of proteins empty vector BMSCs secrete exosome and BMSCs secrete exosome.37 proteins were identified by the above criteria,according to the cell differentiation.The eight proteins involved in cell differentiation.Apoptosis further screening,the six proteins involved in cell apoptosis.Cell differentiation of the protein: Collagen alpha-1(Ⅳ)chain、Slit homolog 2 protein 、 Matrilin-2 、 Connective tissue growth factor 、Phosphatidylethanolamine-binding protein 1、Sulfated glycoprotein 1、Transferrin receptor protein 1、Tenascin.Cell apoptosis proteins: Matrix metalloproteinase-9、Slit homolog 2 protein、Neutrophil gelatinase-associated lipocalin、Connective tissue growth factor 、 Macrophage migration inhibitory factor 、 Peroxiredoxin-5,mitochondrial.Slit homolog 2 protein and Connective tissue growth factor protein are both involved in apoptosis and cell differentiation.Conclusion: Using TMT labeled mouse bone marrow mesenchymal stem cells overexpressing GATA4 secrete exosome analysis about cell differentiation and anti apoptosis proteins.Cell differentiation of the protein: Collagen alpha-1(Ⅳ)chain、Slit homolog 2 protein 、 Matrilin-2 、 Connective tissue growth factor 、Phosphatidylethanolamine-binding protein 1、Sulfated glycoprotein 1、Transferrin receptor protein 1、Tenascin.Cell apoptosis proteins: Matrix metalloproteinase-9、Slit homolog 2 protein、Neutrophil gelatinase-associated lipocalin、Connective tissue growth factor 、 Macrophage migration inhibitory factor 、 Peroxiredoxin-5,mitochondrial.Slit homolog 2 protein and Connective tissue growth factor protein are both involved in apoptosis and cell differentiation.