Role And Mechanism Of Survival Of Heart Transplantation By Exosomes Secreted From Over-expressed IDO In Bone Marrow Mesenchymal Stem Cells

Chapter 1 exosomess secreted by overexpressing IDO rat bone marrow mesenchymal stem cells in the survival of heart transplantationObjective: To detect and compare the regulation of exosomess(BMSCs^IDO-exosomes)secreted by overexpressing IDO rat bone marrow mesenchymal stem cells on DC and T cells in vitro,and to further verify the in vitro results in vivo,trying to prove that BMSCs^IDO-exosomes can effectively improve the heart transplantation survive..Methods: A: BMSCs^IDO-exosomes were co-cultured with DC in vitro.B:Co-culture of BMSCs^IDO-exosomes and T cells.C: BMSCs^IDO-exosomes were co-cultured with DC+T cells.D: BMSCsempty vector-exosomes is co-cultured with DC.E:BMSCsempty vector-exosomes is co-cultured with T cells.F.BMSCsempty vector-exosomes was co-cultured with DC+T cells.G: Co-culture of BMSCs-exosomes and DC.H:BMSCs-exosomes were co-cultured with T cells.I.BMSCs-exosomes were co-cultured with DC+T cells.J: DC are cultivated separately.K: T cells are cultured separately.L: DC and T cells are co-cultured.Flow cytometry was performed at 48 h,72h and 96 h after co-cultivation.Among them,groups A,D,G and J detected CD40,CD86,CD80,MHCII,CD274,CD45 RA,CD45RA + CD45 RB and OX62.Groups B,E,H,and K detected the expression levels of Treg and CD3,CD4,and CD8.The expression levels of CD40,CD86,CD80,MHCII,CD274,CD45 RA,CD45RA +CD45RB,OX62 and Treg,CD4,CD8 were detected in groups C,F,I,and L.2.Simultaneously detect the expression of IDO in groups A,C,D,F,G,I,J and L at 48,72 and 96 hours by RT-PCR.3.Use liquid chromatography to detect IL-1ɑ,IL-4,IL-1β,IL-2,IL-10,IFNr,IL-18,TGFβ1 in groups C,F,I,and L at 48,72,and 96 hours.Changes of TGFβ2 and TGFβ3 factors.In vivo experiment: 2 days,4 days and 7 days after injection of BMSCs^IDO-exosomes,BMSCsempty vector-exosomes,BMSCs-exosomes pre-stained with Dir into heterotopic heart transplantation rats Groups and normal rats were used as the control group.1.Use heart color Doppler ultrasound to detect changes in heart function of the transplanted heart.2.Use the small animal in vivo imaging system to further evaluate the local fluorescence intensity of the transplanted heart.3.Take the spleens of the recipient rats in each group at the above time points,and use flow cytometry to complete the expression of CD40,CD86,CD80,MHCII,CD274,CD45 RA,CD45RA + CD45 RB,and Treg cells.4.Then take the transplanted heart of each group of recipient rats at the above time points,and use HE staining to evaluate the infiltration of inflammatory cells in the transplanted heart.5.Use liquid chromatography to detect IL-1ɑ,IL-4,IL-1β,IL-2,IL-10,IFNr,IL-18,TGFβ1,TGFβ2,TGFβ3 in the serum of the recipient rats of each group at the above time points Factor changes.Results: 1.When co-cultured with DC in each group,it can be seen that at 48,72,and 96 hours,DC surface molecules CD40,CD86,CD80,MHCII,CD45 RA,CD45RA+CD45RB,OX62,BMSCs^IDO-exosomes+DC in the co-culture group expression is reduced,while CD274 expression is increased.The immune cells regulated by IDO are DC.2.When co-cultured with T cells in each group,it can be seen that the number of Treg cells in the BMSCs^IDO-exosomes + T cell culture group in the co-culture group was increased at 48,72,and 96 hours,and there was no significant change in CD4 positive T cells.Regularity,but CD8 positive T cells are reduced,indicating that IDO regulates the T cell population as CD8 positive T cells,and can increase the number of Treg cells.3.Each group was co-cultured with DC+T cells for 48,72,and 96 hours.Visible: DC surface molecules CD40,CD86,CD80,HCII,CD45 RA,CD45RA+CD45RB,OX62 in BMSCs^IDO-exosomes+DC+T cell culture group The expression is reduced,while the expression of CD274 is increased.Again,the immune cell regulated by IDO is DC.The number of Treg cells is increased,and the change of CD4+T cells has no obvious regularity,but CD8+T cells are decreased,which again shows that the population of IDO regulated T cells is CD8+T cells,and can increase the number of Treg cells.4.When co-cultured with DC and DC+T in each group,the expression of IDO was highest in the BMSCs^IDO-exosomes+DC+T cell culture group at 48,72,and 96 hours,and it increased with time.5.When co-cultivating each group with DC+T cells,it can be seen that IL-1ɑ,IL-4,IL-1β,IL-2,IFNr,and IL-18 in the supernatant of each group are reduced at three time points.And there are significant differences with other groups.The expression levels of IL-10,TGFβ1,TGFβ2,and TGFβ3 were increased and were significantly different from other groups.In vivo experiments: 1.After the establishment of the rat heterotopic heart transplantation model,the corresponding treatments were given.It can be seen that the EF and FS of the transplanted heart increased 2 days,4 days and 7 days after treatment with BMSCs^IDO-exosomes and the other groups.2.The small animal in vivo imaging system also shows that the BMSCs^IDO-exosomes group has the strongest local fluorescence intensity in the transplanted heart.3.At 2,4,and 7 days,the expressions of CD40,CD86,CD80,MHCII,CD45 RA,CD45RA+CD45RB in spleen cells of BMSCs^IDO-exosomes group decreased,while the expression of CD274 and Treg cells increased.4.At 2 days,4 days and 7 days,it can be seen that IL-1ɑ,IL-4,IL-1β,IL-2,IFNr,IL-18 in the serum of BMSCs^IDO-exosomes group were reduced and were significantly different from other groups Sexual differences.The expression levels of IL-10,TGFβ1,TGFβ2,and TGFβ3 were increased,and they were significantly different from other groups.5.HE staining showed that the number of inflammatory cell infiltration in BMSCs^IDO-exosomes group was significantly reduced compared with other groups.Conclusion: BMSCs^IDO-exosomes can effectively regulate the surface molecules and inflammatory factors of immune DCs and T cells.Improve the survival of transplanted heart from multiple immune levels.Chapter 2 Detection of BMSCs^IDO-exosomes-micro RNA expression profileObjective: Detection of immune-related micro RAN in BMSCs^IDO-exosomes.Methods: Using illumina small RNA technology to detect BMSCs^IDO-exosomes,BMSCsempty vector-exosomes and BMSCs-exosomes,construct a small RNA library,perform quality inspection on the constructed library,and finally use GO,Pathway and other bioinformatics processing to extract and immune Related micro RNA.Results: The screening criterion was p≤5%,and FC（abs）≥1.5 or ≤0.67 had significant differences,and the following conditions must be met at the same time: 1.It is clear that there is a significant difference between BMSCsempty vector-exosomes and BMSCs-exosomes micro RNA.It is clear that BMSCs^IDO-exosomes and BMSCs-exosomes contain significantly different micro RNA.2.Remove micro RNAs with significant differences between BMSCsempty vector-exosomes and BMSCs-exosomes in micro RNAs containing significant differences between BMSCs^IDO-exosomes and BMSCs-exosomes,regardless of whether the gene is up-regulated or down-regulated.3.After completing the above two conditions,list the top 20 key micro RNAs that are up or down.4.Complete the KEGG and GO enrichment of the top 20 micro RNAs up and down.According to the KEGG analysis,we can see that the upregulated micro RNA shows three pathways according to the genetic prediction results: Intestinal immune network for Ig A production（CD80,AICDA,CD28,ICOS）,Primary immunodeficiency（AICDA,JAK3,ICOS）,Autoimmune thyroid disease（CD80,TSHR,RT1-N2,CD28）.According to KEGG analysis,it can be seen that the down-regulation of micro RNA involves two pathways according to the gene prediction results,namely Intestinal immune network for Ig A production（CXCL12）and Primary immunodeficiency（CD4,RAG2）.According to the genes,there are 7 mi RNAs that are involved in the up-regulation of immunity.Among them,mi R-540-3p has the largest FC increase.Among them,there are 3 genes involved in immune down-regulation,and the FC value of mi R-338-5p has the largest decrease.Therefore,the micro RNAs that we focused on verifying are: up-regulated mi R-540-3p and down-regulated mi R-338-5p.Conclusion: By using illumina small RNA technology to detect immune-related micro RNA in BMSCs^IDO-exosomes,the final up-regulation of key micro RNA was confirmed: mi R-540-3p.The key micro RNA to be down-regulated is: mi R-338-5p.Chapter 3 Quantitative Proteomics Analysis of TMT Markers Related Immunomodulatory Proteins in ExosomesObjective: To analyze the immunomodulatory proteins in BMSCs^IDO-exosomes by TMT labeling quantitative proteomics.Methods: TMT was used to label the proteins contained in BMSCs^IDO-exosomes,BMSCsempty vector-exosomes and BMSCs-exosomes to obtain the original data,and bioinformatics processing such as GO and Pathway was used to extract the relevant immunomodulatory proteins.Results: A total of 1392 proteins were identified in this study,of which 1158 proteins contained quantitative information.If ratio> 1.2 is set,t-test p-value <0.05 is the standard,and the protein is considered to be up-regulated;when ratio <0.83,t-test p-value <0.05 is the standard,and the protein is considered to be down-regulated.Among the quantified proteins,317 proteins were up-regulated and 114 proteins were down-regulated in BMSCs^IDO-exosomes compared with BMSCs-exosomes.A total of335 proteins were up-regulated and 108 proteins were down-regulated in BMSCs^IDO-exosomes compared to BMSCsempty vector-exosomes.A total of 34 proteins were up-regulated and 43 proteins were down-regulated in the BMSCsempty vector-exosomes than the exosomes group secreted by BMSCs.Based on the above data,the following screening criteria were further adopted: 1.BMSCsempty vector-exosomes compared to BMSCs-exosomes comparison group as background,using BMSCs^IDO-exosomes to BMSCs-exosomes group and BMSCs^IDO-exosomes to BMSCs empty vector-exosomes group Different proteins with the same change trend in the comparison group of BMSCs-exosomes and BMSCsempty vector-exosomes were eliminated.2.Remove the proteins with different trends in BMSCs^IDO-exosomes than BMSCs-exosomes group and BMSCs^IDO-exosomes than BMSCsempty vector-exosomes group.3.Proteins that meet the conditions of 1 and 2 are analyzed according to 1.2times.Two methods are used: 1.List the top 20 proteins（up 20 and down 20）.2.According to KEGG Pathway,screen out the proteins that meet the conditions of 1 and 2.Among them,there are 5 immune-related proteins,which are: Transforming protein Rho A,Mitogen-activated protein kinase 1,Cell division control protein 42 homolog,Tyrosine-protein phosphatase non-receptor type 11,Four and a half LIM domains 1.The intersection of the two methods shows that the key protein is Four and a half LIM domains 1.Conclusion: The key protein involved in immune regulation in BMSCs^IDO-exosomes was identified as FHL-1 by using TMT to label BMSCs^IDO-exosomes protein.