| Objective:Human umbilical cord mesenchymal stem cells (hUC-MSCs) were often used in cell therapy for myocardial infarction. We assumed that hypoxic preconditioning of hUC-MSCs (HP-MSCs) could enhance their ability of anti-apoptosis and paracrine. In this study, hUC-MSCs was isolated and expanded, and preconditioned with hypoxia in different oxygen concentration. We observed the changes of capacity for anti-apoptosis and paracrine of HP-MSCs. The HP-MSCs were then transplanted into a mini swine myocardial infarction mode to investigate the effect of cell treatment.Methods:The hUC-MSCs were cultured and expanded to passage6, and were treated in hypoxic incubator with different oxygen concentration for24h before the test. hUC-MSCs was divided into5groups according to the oxygen concentration of hypoxic preconditioning:1%O2,3%O2,5%O2,10%O2,21%O2(control), In vitro, FACS, RT-qPCR, ELISA and Annexin V/PI were performed to investigate the gene expression and cytokine secretion, and the ability of anti-apoptosis of HP-MSCs. Transplantation of HP-MSCs into infarct area was applied surgically on the Zhonghua mini swine. The swine were randomly divided into three groups (n=6in each): Control (normal saline injection), Nor (normal hUC-MSCs injection) and Hyp (HP-MSCs injection). About2.5x107cells in3ml were injected into peri-myocardial infarction area in15spots. Echocardiogram and Single-Photon Emission Computed Tomography (SPECT) were performed at immediately and six weeks postoperatively respectively.6weeks after operation, the animals were euthanized and myocardium tissue in peri-MI area of the heart was harvested to analyze the engraftment of stem cells, myocardium apoptosis and angiogenesis by Masson trichrome staining, immunofluorescence and TUNEL staining.Results:(1) The phenotype of hUC-MSCs were positive in CD73、CD90、CD105and negative in CD11b/CD34/CD45/CD19and HLA-DR. RT-qPCR indicated that the gene expression of Bax in1%O2and3%O2groups were down-regulated significantly compared with control and10%O2groups (p<0.01); The gene expression of Bcl-2in1%O2,3%O2,5%O2and10%O2groups were up-regulated significantly compared with control group (p<0.01); After apoptosis induction, Annexin V/PI test demonstrated that the percentage of apoptotic cells in all the hypoxic preconditioning groups decreased significantly as compared to positive control group (p<0.01).RT-qPCR showed that the gene expression of HGF in1%O2,3%O2and5%O2groups were up-regulated significantly as compared to control group (p<0.01). VEGF gene expression in3%O2and5%O2groups increased significantly compared with every other groups (p<0.01). NGF gene expression in3%O2and5%O2groups increased as compared to the control group (p<0.01). The results of ELISA revealed that the protein secretion of HGF in1%O2and3%O2groups raised significantly compared with control and10%O2groups (p<0.01). The protein of VEGF in3%O2group increased significantly as compared to the control and10%O2groups (p<0.01). The protein of KGF in3%O2and5%O2groups increased significantly as compared to the control and1%O2groups (p<0.01). The protein of NGF in3%O2and5%O2groups increased significantly as compared to the control group (p<0.05)(2)6weeks after transplantation, echocardiogram show that LVEDV in Hyp group reduced significantly as compared to the control group (p<0.05). The change of LVEF in Hyp group improved compared with control group (p<0.05). AWT%in cells treatment groups reduced as compared to the control group (p<0.01) SPECT revealed that AMDP in the cells treatment groups decreased compared with the control group (p<0.01).The change of LVEF in Hyp group increased as compared to the control group(p<0.05). Masson trichrome staining analysis show an improvement in fibrosis area in Hyp group as compared to the control group (p<0.05).(3) β-microglobin was positive only in human cells, which means it reflect the engraftment of human MSCs transplanted into the heart of swine. More positive cells were catched in Hyp group compared with the Nor group (p<0.05). TUNEL staining show that more apoptotic cells were observed in control group as compared to cell treatment group (p<0.01). Isolactin B4is a marker of vascular endothelial cells. More Isolactin B4postive staining were found in Hyp group as compared to the control group(p<0.01). Conclusions:(1) Hypoxic preconditioning hUC-MSCs in3%oxygen concentration enhanced their ability of anti-apoptosis and secretion of cytokines.(2) Transplantation of HP-MSCs improved the restoration of heart function in some extent. (3) HP-MSCs could survive better in vivo, reduce the apoptosis of cells in the heart, enhance the angiogenesis and reduce the scar by paracrine effect. |
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