| Heart failure,is a great challenge to global clinical and public health at present,with high morbidity and mortality.The latest cardiovascular disease report in China shows that the prevalence of heart failure in China is 0.9%,with an incidence of 0.7‰ to 0.9‰.By 2018,there are 4.5million patients with heart failure in China.The drug treatment of heart failure has been standardized,and the instrument treatment is more advanced,but the hospitalization rate and mortality rate of heart failure patients are still high.In recent years,research on stem cells and microvesicles(MVs)has become hot.Many evidences suggest that stem cells and MVs can play a certain role in improving cardiac function,promoting angiogenesis and promoting cell regeneration.All subtypes of cells can release MVs during activation or apoptosis,which play a role in signal transmission,intercellular communication and tissue remodeling.The preliminary study and literature retrieval in this study suggested that myocardial remodeling in the process of heart failure may be related to MVs,and further discovery of the mechanism of MVs in the pathological process of heart failure may provide a fundamental new idea for the treatment of myocardial remodeling.We intend to analyze the basic characteristics of MVs in circulating blood of patients with heart failure.The effects of circulating blood MVs on heart failure were studied by comparing and analyzing the changes of MVs protein and mi RNA in circulating blood of patients with heart failure and healthy subjects.Plasma was extracted from the subjects of heart failure group and healthy control group,and MVs were extracted by exclusion + superion method.The MVs were identified by surface marker proteins detected by Western Blot(WB).The qualified MVs were analyzed by proteomics and mi RNA to obtain differentially expressed proteins and mi RNAs.Finally,myocardial cells and MVs were co-cultured to verify the effect of MVs.There were no significant differences in age,body weight and gender between the two groups,but there were significant differences in cardiac Ejection Fraction(EF)and Brain Natriuretic Peptide(BNP).The concentration of MVs in heart failure group was 5.7×1010 MVs per ml,and the particle size peak was 93.2nm,while the concentration of MVs in healthy group was 1.5×1010 MVs per ml,and the particle size peak was129.1nm.The concentration of MVs in heart failure group was significantly higher than that in healthy control group,but the particle size was smaller than that in healthy control group(P < 0.05).The morphology of MVs was observed under transmission electron microscope.MVs in the healthy control group and the heart failure group were in the shape of homogeneous teacups without significant difference in morphology.WB showed that the positive markers of Tsg101,CD63 and CD9 typical MVs were expressed in both groups,and the negative marker Calnexin was expressed.It was verified that the extract was MVs,which met the international standards of MVs.There were 310 kinds of proteins in the heart failure group and 218 kinds of proteins in the healthy control group.A total of 21 kinds of proteins were found by statistical analysis of differential proteins in the two groups,including 8 kinds of differential proteins up-regulated and 13 kinds of differential proteins down-regulated.Differential expression protein Gene Ontology(GO)annotation classification statistics showed that: Biological processes such as complement activation,complement activation regulation,recognition phagocytosis,neutrophil degranulation,and other cellular components such as MVs,extracellular space,outer plasma membrane,blood particles,antigen binding and immunoglobulin binding molecular functions are significantly different in proportion.The number of proteins annotated to complement and clotting cascade,dilated cardiomyopathy,the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways is significant;There were 23 differentially expressed mi RNAs in the two groups,among which 14 differentially expressed mi RNAs were up-regulated and 9differentially expressed mi RNAs were down-regulated.Eight heart failure related mi RNAs were annotated in HMDD database,including 3up-regulated mi RNAs and 5 down-regulated mi RNAs.Red PKH26 labeled MVs.After adherence,the myocardial cells were co-cultured with peripheral blood MVs of the heart failure group and the healthy control group for 12 hours,respectively.The nuclei were labeled with blue Hochest,and the cytoskeleton was labeled with green ghost ring peptide,and the results showed that AC16 cells internalized MVs.After cell adhesion and co-culture with 50μg MVs for 24 h,the cell state of the heart failure group was significantly worse than that of the control group under ordinary microscope,and the cells showed a tendency of apoptosis.Further quantitative detection of cell viability showed that the activity of myocardial cells in the MVs treatment group was significantly decreased in the heart failure group.The expression of apoptosis-related protein in cardiomyocytes was detected by ELISA after the co-culture of cardiomyocytes with the two groups of MVs.The results showed that the apoptosis-related protein caspase9 in cardiomyocytes was significantly increased after the interaction of MVs in the heart failure group.Based on the above study,we can draw the following three conclusions :1.MVs can be isolated and extracted from circulating blood of heart failure patients,and the number of MVs is increased compared with healthy control group 2.Circulating blood MVs are involved in the progression of heart failure by participating in inflammatory responses such as apoptosis of cardiomyocytes.MVs in circulating blood mediate the development of heart failure through their contents and are important mediators in the pathological process of heart failure. |
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