| Rationale:Due to the heart’s limited self-regeneration capacity after myocardial infarction(MI),additional strategies are needed to enhance cardiac repair in patients with MI.Many studies have suggested the effectiveness of mesenchymal stem cells(MSCs)therapies in MI.However,the biggest barrier to successful stem cell therapies is the poor cell homing.Cytoprotective strategies to enhance donor cell homing are critical requirement for successful cardiac repair.One attractive approach may be the combination of cell-and gene therapy.Genetically modifying MSCs to overexpress a protein which can improve cell homing may yield greater benefits.Integrin-linked kinase(ILK),a widely expressed serine/threonine protein kinase,is highly expressed in the heart.ILK closely related to key molecules for MSCs homing via binding to the cytoplasmic domain of p-integrins.We have demonstrated that ILK gene can regulate cell growth,survival,proliferation,as well as differentiation.But the effect of ILK on MSCs homing is unclear.To achieve this goal,we intend to transplant the ILK transfected MSCs after labeling SPIO into swine MI model,and then dynamically monitor MSCs migration,homing and effect on cardiac function using MR.Hopefully,this project will offer new ideas and scientific basis for improving clinical efficiency of MSCs-basedtherapy.Methods:1.We isolated MSCs from swine bone marrow.After genetically modified with adenovirus containing GFP/ILK or GFP only,we assessed cell viability,migration ability,proliferation rate and cell apoptosis level in vitro.2.MSCs was labeled with ultrasmall superparamagnetic iron oxide(USPIO)before transplantation and then performed MR molecular imaging.we assessed the effect of the USPIO on cell viability,migration ability,proliferation rate and cell apoptosis in vitro.3.PBS or MSCs or ILK.MSCs(5×107 cells)were randomly transplanted into the ischemic myocardium via coronary artery 1 week after establishing the swine myocardial infarction model by ballon occlusion.The cell cardiac homing was monitored at 24h,3d,lw and 2w after transplantation using MR molecular imaging.The myocardial blood perfusion was assessed by MR first pass perfusion.The infarction area was evaluated by delayed enhanced examination.The GFP expression was calculated from frozen section.The USPIO in transplanted cells was detected by prussian blue staining.The cardiac fibrosis,capillary density,cell apoptosis and cell proliferation were assessed using immunohistochemistry two weeks later.4.The data was statistically analyzed with Independent Sample t test for two group comparison and ANOVA test for three group comparison using SPSS 17.0 software.Results:1.Over-expression of ILK increased the viability(p<0.001),proliferation rate(p<0.001),and migration ability(p<0.001)of MSCs as well as reduced cell apoptosis(p<0.001)in vitro.2.The USPIO labeling rate of MSCs was 100%.The optimal labeling concentration was 50ug/ml in our study.the minimum detectable cells number was 1×104/0.5ml cells.The MR signal intensity was decreased by the increasing of labeling concentration,the cell numbers and the cell passages.3.In vivo MR molecular imaging manifested in MSCs and ILK-MSCs Groups that the hypointensity area and signal intensity variation at 3d were larger/higher than those at 24h(p<0.05);the area was increased(p<0.05),but the intensity variation was decreased compared with that at 3d(p<0.05);the area and intensity variation were all decreased 2 weeks later compared to those at lweek(p<0.05).The area and intensity variation of ILK-MSCs group was larger/higher in former 3 time points compared with those of MSCs group(p<0.05),but smaller/lower in 2weeks(p<0.05).Transplantation of ILK-MSCs improved myocardial perfusion,decreased infarction area and improved reginal eardiac function(p<0.05).More GFP positive and prussian blue staining positive cells were found in ILK-MSCs group two weeks after cell transplantation(p<0.001),ILK-MSCs group also showed increased angiogenesis(p<0.001)and proliferation and reduced fibrosis(p<0.001)and cell apoptosis(p<0.001)in the peri-infarct myocardium.Conclusion:Transplantation of ILK.MSCs after myocardial infarction can increase the homing rate of MSCs.It can effectively improve the regeneration capacity of MSCs as well as the cardiac repairing character of ILK gene.Our study showed increased angiogenesis and proliferation and reduced fibrosis and cell apoptosis in the peri-infarct myocardium after implantaton of ILK-MSCs,and thus lead to improvement of the curative effect of swine myocardial infarction.Our results will offer new ideas and scientific basis for improving clinical efficiebcy of MSCs-based therapy. |
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