| Background:Acute myocardial infarction(AMI)is one of the common clinical disease with high rate of mortality.Currently,common therapy cannot compensate for substantial damage caused by AMI.Human umbilical cord derived mesenchymal stem cells(hUC-MSCs)have showed wide clinical application prospect in tissue engineering,because of their collecting easiness and low immunogenicity.Early studies suggested that hUC-MSCs could proliferate and differentiate into myocardial cells to compensate for the damaged tissue in the treatment of myocardial infarction,but later studies pointed out that there had no myocardial cell differentiated from hUC-MSCs which could be detected in the myocardial tissue,but factors associated with the angiogenesis changed,which suggested that hUC-MSCs mainly treat myocardial infarction through paracrine.In recent years,exosomes derived from stem cells which have definite therapeutic effect on myocardial infarction have been considered a key factor for stem cell therapy,and studies have shown that P4 hUC-MSCs had the best curative effect on myocardial infarction,while from P7 hUC-MSCs,the therapeutic effect became worse,and the rate of proliferation and morphology got changed.It is considered that the generation change of hUC-MSC may affects its paracrine mechanism then further affects the curative effect of stem cells on myocardial infarction.Therefore,in order to clarify whether exosomes play an important role in the efficacy of hUC-MSCs,and to further explore whether the efficacy of myocardial infarction could be affected by paracrine mechanism of the different generation of hUC-MSCs,we chose exosomes from P4 and P7 hUC-MSCs in this study to conduct animal experiments and to observe their efficacy of myocardial infarction,then we further compared their protein content and miRNA,in order to provide a scientific basis for further exploration of the mechanism of exosomes in the treatment of myocardial infarction.Objective:To study the different therapeutic effect of exosomes derived from different generation of hUC-MSCs on myocardial infarction.Methods:1.The human umbilical cord mesenchymal stem cells were obtained by tissue block method.The morphology were observed under microscope,the surface markers were identified by flow cytometry and the multidirectional differentiation of adipogenesis and osteogenesis were also detected.2.Exosomes from mesenchymal stem cells were extracted by ultra speed differential centrifugation,Exo-quick Kit and Isolation Kit.The particle sizes,morphological identification,protein concentration,protein compositions and marker protein CD9,CD63 and CD81 of exosomes extracted by three methods were detected by particle size analyzer,transmission electron microscope,BCA method,SDS-PAGE gel electrophoresis,Western blot respectively.3.Exosomes of P4 and P7 mesenchyme stem cells were extracted by superspeed centrifugation,then comparing the partical sizes,morphology,protein concentrations,the expression of marker proteins and miRNA21 content of them.4.Myocardial infarction(MI)models were established by ligating the left anterior descending coronary artery in rats.The experimental groups were injected with equal volume exosomes from P4 and P7 cells,while the control group was injected with the same volume NS.Myocardial cell apoptosis was detected by echocardiography,myocardial tissue TTC staining and Western blot.The differences of cardiac function between infarction group and two treatment groups were compared.Results:1.The P4 hUC-MSCs was arranged in a tight spiral pattern like hair under low magnification,and spindle in parallel under high magnification.The P7 hUC-MSCs were in a disorderly arrangement compared with the P4 cells.Flow cytometry showed that all two generation cells were strongly positive for CD90,CD44 and CD105,and CD14,CD34 and CD45 were all negative.There was no significant difference in the expression rate(P > 0.05).The P4 and P7 cells could be successfully induced to adipocytes and osteoblasts.2.Exosomes were extracted by all three methods.Under the electron microscope,circular or elliptical membranous vesicles can be seen from 30 to 200 nm;Through particle size analyzer,the diameter of vesicles got by the superspeed differential centrifugation,Exo-Quick Kit,Isolation Kit were 86.65+0.96 nm,187.95+3.22 nm,77.55+1.29 nm;The Exosomes marker protein CD9,CD63 and CD81 were all positive in the three methods,there was significant difference in the expression rate(P<0.01,P<0.05);protein concentrations were 2.78+0.13mg/ml,2.14+0.13mg/ml,1.38+0.11mg/ml,There was significant difference in the expression rate(P<0.05),and the differences were obvious;protein is mainly concentrated in 25-55 kD and over 130 kD,and there were differences in stripes.3.The AMI model of rats were successfully established.Exosomes from hUC-MSCs were injected into myocardium of AMI rats after 7 days,exosomes of P4 hUC-MSCs were called the P4/Exo,and exosomes of P7 hUC-MSCs were called the P7/Exo.Ultrasonography showed that LVIDd and LVIDs of AMI+NS group were higher than that of AMI+P4/Exo group and AMI+P7/Exo group,and the differences of three groups were statistically significant(P < 0.01).EF and FS of AMI+Sham group were both decreased compared with that of AMI+P4/Exo group and AMI+P7/Exo group,and the differences of three groups were statistically significant(P < 0.01).Myocardial infarction sizes measured by TTC were different in all groups,and there were statistical differences(P < 0.05).Apoptosis was detected by flow cytometry,the apoptotic rate in Sham group,AMI+NS group,AMI+ P4/Exo group and AMI+P7/Exo group were 3.61±0.74%,46.77±0.51%,26.39±0.92%,35.38±1.37%,respectively,and the differences were statistically significant(P < 0.05).Western blot detection of apoptotic proteins showed that the expression of Caspase-3 in AMI group was higher than that in AMI+P7/Exo group,the expression in AMI+P7/Exo group was higher than that in AMI+P4/Exo group,and the expression in AMI+P4/Exo group was higher than that in Sham group,while the expression of Bcl-2 was the opposite.4.Exosomes from P4 and P7 hUC-MSCs were extracted by superspeed differential centrifugation.Under transmission electron microscopy,uniform,pallet-like elliptic vesicles were observed;particle size were 98.33 + 7.65 nm,104.68 + 6.68 nm respectively;the protein concentration which were measured by BCA method were 2.64 + 0.12 g/ L,2.45 + 0.13 g/ L respectively,and the difference was not statistically significant;the related marker protein CD9 and CD63 were all positive;the relative expression of miRNA21 of exosomes derived from P7 hUC-MSCs was 0.89 + 0.04 with miRNA21 of exosomes from P4 hUC-MSCs as a parameter,there was statistically significant difference in t test analysis with independent samples.Conclusion:1.Compared with the three methods,the quality of exosomes extracted by superspeed differential centrifugation was the best.2.The AMI model of rats were successfully established.Exosomes from P4 hUC-MSCs had better curative effect on myocardial infarction compared to those from P7 hUC-MSCs,which suggested that exosomes play an important role in the therapeutic effect of hUC-MSCs.3.The compare between exosomes from P4 and P7 hUC-MSCs has shown that the change of mi RNA21 was statistically significant,which may suggested that exosomes could play a role through miRNA21. |
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