| BACKGROUNDMesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal capacity. Human umbilical cord mesenchymal stem cells(hUC-MSCs) have become an ideal stem cell for clinical transplantation due to its rich content, lower immunogenicity, no injury to donors when collected, ethics unvolved and good therapeutic effects on tissue repair and regeneration.Although hUC-MSCs have good potential for its unique biological characteristics, there are still much to be done. For example, hUM-MSCs lack specific cell surface markers, preservation procedure before transplantation has not been standardized. Besides, limitations exsist for clinical application of hUC-MSCs because of its the uncontrollable immune regulation, potentially tumorigenic property, non-therapeutic differentiation and abnormal accumulation. In addition, the proposal of "paracrine" mechanism of MSCs makes the study of MSCs increasingly important. A cell membrane vesicle-Exosomes has become the focus of research in the field. Many cells can secrete Exosomes, a complex subcellular structure, which is considered to transfer information between cells, mediating important "endocrine" mechanism. Compared with other cellular secreting vesicles, Exosomes have clearer biological properties. Optimizing the preparation technique of Exosomes, exploring its potential therapeutic effects compared to its deriving hUC-MSCs and revealing the core mechanism of MSC may provide a foundation for further exploring a brandnew type of stem cell therapy as "no cell therapy".Objectives1.Exploring optimized preparation technique of hUC-MSCs derived Exosomes;2.Comparative Study on therapeutic effects of hUC-MSCs and their Exosomes in vivo;3.Revealing the core mechanism of stem cell treatment and providing scientific basis for further exploring the "no cell" stem cell therapy.Methodsl.hUC-MSCs were isolated and cultured using adherent tissue block method with internationally recognized standards. Comprehensive identification morphology, surface markers and multipotent differentiation capacity of hUC-MSCs were detected; 2.hUC-MSCs derived Exosomes were prepared using a modified differential centrifugation method. Morphological identification were done by TEM line and protein levels were measured by Bradford. Furthmore, protein components were preliminary analyzed by SDS-PAGE gel electrophoresis and surface marker, CD63, CD81were detected by Western blot assay;3.Rat model of myocardial infarction were established by ligation of the left anterior descending coronary artery. Rats were randomly divided into4groups (n=15):①﹕ham+NS group (only threading the left anterior descending artery without ligation and,0.9%sodium chloride was injected);②MI+NS group (0.9%sodium chloride was injected);③MI+MSCs grou (After the digestion of hUC-MSCs, cells were resuspented with0.9%sodium chloride injection and concentration of cells was adjusted to2×106cells/mL.80μL of the solution was injected);④MI+Exosomes group (80μL of Exosomes containing80μg protein was injested). The transplantation therapy was given after2weeks of the model establishment;4.Echocardiography for each rat was underwent before model establishment, after2weeks of modeling and4weeks of transplantation therapy to observe the changes of cardiac function;5.Infarct size was measured using TTC staining after4weeks of transplantation;6.Apoptosis of myocardial cells in left ventricular myocardial tissue was detected by flow cytometry after4weeks of transplantation;7.Expressions of myocardial apoptosis related proteins Bcl-2and Caspase-3were tested by Western blot after4weeks of transplantation.Results1.The cultured hUC-MSCs through tissue culture block method meet the accreditation standards;2.The prepared hUC-MSCs derived Exosomes through differential centrifugation method was observed under TEM which showed that the diameter was30～100nm, membrane was intact, intracellular components was with low electron density, vesicular structure was round or oval and CD63and CD81were expressed. Protein levels from106/hUC-MSCs cells was11.63±0.445μg;3.Exosomes and hUC-MSCs were transplanted into rats by epicardial injection method. Heart ultrasound results showed that:the difference of the parameters was not statistically significant (p>0.05) between groups before modeling; after modeling parameters were not significantly different between2weeks group and the group before modeling and also between sham+NS groups (p>0.05). In the rest of the groups, LVIDs and LVIDd were significantly increased, and EF and FS were significantly decreased (p<0.001). Parameters were not significantly different between groups after4weeks of transplantation and group after2weeks of modeling and also between sham+NS group and MI+NS group (p>0.05). LVIDd and LVIDs are reduced in the transplantation group. Besides, the EF and FS were increased, the difference was statistically significant (p<0.001). Parameters between MI+Exosomes group and MI+MSCs group was statistically significant difference(p<0.01) except the LVIDs. The rest of the parameters between the two groups was not statistically significant (p>0.05);4.TTC results showed that in sham+NS group, there is no infarct area compared with MI+NS group (42.10±1.181%). In hUC-MSCs transplantation group and Exosomes transplantation group, the infarct size was significantly reduced (31.47±0.625%) vs (33.53+0.737%), the difference was significant (p<0.001). However, there is no significant difference between MI+MSCs group and MI+Exosomes group (p>0.05);5.Flow cytometry showed that apoptosis rates were significantly decreased in MI+MSCs group (21.69±2.768%) and MI+Exosomes group (20.86±2.729%) compared with MI+NS group (36.39±1.038%)(p<0.001). But no significant difference between MI+MSCs group and MI+Exosomes group was observed (p>0.05);6.Western blot results showed that expressions of Bcl-2and Caspase-3in myocardial tissue in MI+NS group was significantly increased compared with sham+NS group (p<0.001). The Bcl-2protein expression was increased and the Caspase-3protein was decreased after hUC-MSCs or Exosomes transplantation compared with MI+NS group, the difference was significant (p<0.001). However, between MI+MSCs group and MI+Exosomes group, there is no statistically significance (p>0.05).Conclusions1.hUC-MSCs derived Exosomes prepared with differential centrifugation technique has good purity which provides a stable and reliable source for subsequent in vivo transplantation experiments;2.hUC-MSCs and Exosomes can significantly improve left ventricular function reduce infarct size and inhibit apoptosis of myocardial cells after myocardial infarction of rats. The anti-apoptotic effect of both may be one of the important mechanisms to delay the progression of myocardial infarction;3.No significant differences were found in the therapeutic effect between hUC-MSCs and Exosomes on myocardial infarction. |
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