The Effects Of Self-assembling Peptide Nanofibers Scaffold On The Survival, Differentiation Of MCSCs And Cardiac Repair

Objective:Explore the effect of diazoxide(DZ) preconditioning on bone marrow-derived cardiac stem cells(MCSCs) under oxidative stress and its mechanism. Explore the effect of the self-assemble peptide(SAP) nanofiber scaffold(NFS) on the proliferation,survival and differentiation of MCSCs.Aim to provide theoretical and experimental basis for the new nano-material SAP used in clinical stem cell transplantation for treatment of ischemic heart disease such as myocardial infarction (MI).Methods:MCSCs were selected from marrow mesenchymal stem cells with single-cell cloning culture,c-kit immunoflourence staining and Nkx2.5 expression. The oxidative stress of MCSCs was caused by H₂O₂.With Annexin V/PI double staining and lactate dehydrogenase(LDH) leakage,it was detected the effect of DZ preconditioning on MCSCs under oxidative stress.The application of cytochrome C staining,RT-PCR and Western blot to explore the mechanism of the effect.The self-assemble peptides RAD-â…¡were designed and synthesized.The self-assemble state was viewed by AFM and SEM.cell suspension was mixed with equal volume of 1% RAD-â…¡.After incubation for one day,the compatibility of MCSCs with self-assembling peptide nanofiber scarffolds was viewed by SEM.The effect of NFS on the growth and survival of MCSCs was detected by CCK-8 and AO/EB staining. Through cardiac-specific troponin cTnT staining,the effect of NFS was evaluated on myocardial cell differentiation of MCSCs.Thirty minutes after the establishment of acute myocardial infarction model of female rats,the rats were injected at the edge of infarcted area with PBS,MCSCs,pretreatment MCSCs,SAP+MCSCs,SAP+ pretreatment MCSCs respectively.Four weeks after MI,the cardiac function change was detected by M-mode echocardiography,the change of organizational structure was detected by masson staining,the microvessel of the surrounding scar tissue marked with vWF stained.The donor cells in myocardial tissue were markered with Y chromosome through Fluorescence in situ hybridization.The expression of cTnT of Y chromosome-positive cells was detected by Immunohistochemical.Results:There was a significantly increase of the number of viable cells and reduce of LDH leakage in DZ group compared with H₂O₂ group.After oxidative stress injury,cytochrome C translocation from mitochondria to the cytoplasm of cells.In the DZ group,the cells of cytochrome C punctate staining are significantly more than H₂O₂ group.HGF and bFGF expression and phosphorylated Akt of the cells was significantly raised under oxidative stress after pretreatment with DZ.SEM show the peptides self-assembly into a mesh of interwoven nanofiber scaffold structure,the stent diameter was 50 200 nm.In vitro studies,MCSCs cultured in the self-assemble peptide nanofibers 1 day later,cells located in the network-like nanofiber and closely linked with the nano-fibers under SEM.The network-like nano-fibers around the cell and closely linked with the cells under TEM.The viability of cells in NFS was significantly higher than that of control group.The difference of apoptotic cells was significant after 8 hours between the NFS and control groups,when MCSCs cultivante with sugar-and serum-free DMEM under the hypoxia condition.The cells in NFS connect closely each other with the same direction and arrangement neatly,all of the cells express cTnT at 4 weeks after induction.In four weeks after cell transplantation on rats of MI,Functional recovery of rat hearts,myocardial scar size reduction and the Y chromosome-positive cells of the pretreatment MCSCs transplantation group are superior to MCSCs transplant group,the difference was significant.Compared with the pretreatment MCSCs transplantation group,in the two cell transplantation using the SAP groups,heart function index markedly improved,scar area significantly reduced,marked increase in microvessel density,Y chromosome-positive cells increased significantly,the differences were very statistically significant.The differences were not statistically significant between the SAP+MCSCs group and SAP+pretreatment MCSCs group.The distribution of Y-chromosome positive cells are concentrated of the two cell transplantation using the SAP groups.Y chromosome-positive cells expressed cTnT.Conclusions:MCSCs pretreatment with DZ can reduce cell oxidative stress damage,which may be related to paracrine improvement and reduce mitochondrial cytochrome C translocation to the cytoplasm. NFS and MCSCs have good biocompatibility.NFS can build an three-dimensional micro-environment conducive to MCSCs growth,survival and differentiation.The injectable self-assemble peptide build three-dimensional environment is conducive to the survival and towards functional myocardial differentiation of MCSCs in the ischemic myocardium,and induce angiogenesis,a significant reduction in scar area and improve cardiac function.Its efficacy is superior to DZ pretreatment stem cell transplantation.This study provide theoretical and experimental basis for the new nano-material SAP used in clinical stem cell transplantation for treatment of ischemic heart disease such as myocardial infarction.