| Background:Mesenchymal stem cells(MSCs)are a kind of pluripotent stem cells with self-renewal ability,which can differentiate into osteoblasts,adipose cells,endothelial cells,smooth muscle cells and other cell types under certain induction factors,and MSCs can secrete various growth factors to induce angiogenesis.Therefore,MSCsis a prospective candidate in regenerative medicine.Currently,ischemic diseases therapy with stem cell transplantation is developing rapidly,and its safety and effectiveness have been confirmed by different studies.MSCs treat ischemic cardiomyopathy by transplanting or mobilizing stem cells into ischemic myocardial tissue,and increasing the number of myocardial cells and capillaries in infarcted areas,reversing cardiac remodeling,and repairing or strengthening the biological functions the heart.However,the ability of MSCs to differentiate into cardiomyocytes is still controversial,but the capability of MSCs differentiate into endothelial cells,smooth muscle cells or peripheral cells has been confirmed.MSCs transplantation could promotes myocardial repair by stimulating angiogenesis,and this make them as a promising cell type in ischemic heart disease therapy.Hedgehog(Hh)is a morphogen which play an important role in embryonic development,and regulating the tissues patterns during embryonic development.There are three homologous genes of Hedgehog in mammals:Shh,Ihh and Dhh,and Shh is the most studied member of Hh family.VEGF signaling pathway is one of the most important signaling pathways to regulate vascular development and endothelial cell differentiation.To date,the mechanism of VEGF inducing MSCs to differentiate into endothelial cells has not been clarified.Shh is related to many angiogenic factors,such as promoting the expression of VEGF and participating in the growth of blood vessels,and has a significant therapeutic effect on acute or chronic myocardial ischemia model and ischemia reperfusion injury model.Shh can activate vascular endothelial growth factor(VEGF)and angiopoietin-two important growth factors in angiogenesis,so Shh may be one of the keyregulatory molecules for angiogenesis and maybe an attractive therapeutic tool for enhancing angiogenesis.Part I Construction of rat BMSCs with overexpression of Shh geneObjective:Isolated mesenchymal stem cells from ratsbone marrow,and overexpressed Shh in MSCs by lentivirus system.Method:Bone marrow mesenchymal stem cells(BMSCs)were isolated from young rats,and the Passage 3 BMSCs were detected by FCM.The Shh gene was successful amplificated by PCR,and overexpressed in BMSCs by lentivirus system.Results:The results showed that CD90 and CD29 antigens were highly expressed on the cell surface,while MHC Ⅱ and CD11b/c were not expressed,indicating that the cells we isolated and cultured were bone marrow mesenchymal stem cells.RT-qPCR detection found that the expression of Shh mRNA was increased in MSCShh,and Shh expression was increased by about 3000-fold and 5000-fold at three days-transfection and seven days-transfection,respectively.Conclusion:BMSCs were successfully isolated from rats and established Shh-overexpressed MSCs that would be a cell model for studying the effects of Shh on BMSCs to endothelial differentiationPart Ⅱ Shh gene promotes BMSCs differentiated intoendothelial cellsObjective:To investigate the effect of overexpression of Shh gene on the differentiation of MSCs into endothelial cells in vitro.Method:RT-qPCR detected the angiogenic molecules,such as Patched 1,insulin-like growth factor 1,hepatocyte growth factor,angiopoietin-1 and VEGF-A mRNA levels in MSCShh cells after Shh transfection for 3 days and 7 days.The capacity of angiogenesis and migration were accessed by tube formation assay and transwell assay.Results:After overexpression of Shh,the ability of tube formation,migration and differentiation into endothelial cells of MSCs were significantly higher than control group.Tube formation assay found that the tube length and mashes numbers were significantly higher at 7 days after transfection than that at 3 days after transfection,and the results of endothelial differentiation of MSCshh was consistent with the tube formation assay.Conclusion:Angiogenesis of BMSCs is enhanced after overexpression of Shh,which indicates that Shh plays an important role in regulating endothelial differentiation of mesenchymal stem cells.Part Ⅲ Effects of MSCShh in cardiac repair after myocardial infarction in ratsObjective:To investigate the cardiac repair effects of MSCShh in rat myocardial infarction model.Method:MSCs were transplanted into rat myocardial infarction model with or without Shh overexpression.Echocardiography was performed to detect the recovery of cardiac function,and Masson staining was used to access the fibrotic area after myocardial infarction.Results:Both MSCNC and MSCshh were able to improve cardiac function after myocardial infarction,and the repair effect of MSCShh group was more obvious than that of MSCNC group.Conclusion:After overexpression of Shh gene,MSC cells can more effectively reduce infarct area and improve cardiac function.Part Ⅳ Discussion on the mechanism of Shh gene promotes BMSCs differentiation into endothelial cellsObjective:To further explore the specific mechanism of MSCShhdifferentiation intoendothelial cells.Method:Both of MSCNC and MSCShhwere carried out high-throughput sequencing,and the sequencing results were preliminarily analyzed to identify different expressed genes and their target genes.MSCShh with VEGF-D siRNA transfection transplanted into the rat acute myocardial infarction model,and echocardiographic detected the restore of cardiac function at 3 days,7 days,14 days and 28 days after surgery.Results:The results showed in MSCShh there are 330 differentially-expressed genes were detected,138 genes were up-regulated and 192 genes were down-regulated compared with MSCNC.Interestingly,the expression of genes related with angiogenesis in MSCShh,such as VEGF-D,Angptl4,Egf16,Pde7b,Amigo2 and Ednrb,were much higher than those in MSCNC.It was found that the expression level of VEGF-D was significantly upregulated after overexpression of Shh.After transfecting MSCShh with VEGF-D siRNA,the cells presented attenuated tube formation ability,the tube length and mashes numbers were significantly decreased.Compared with the PBS control group,MSC and MSCShh can improve the heart function after myocardial infarction,however,the restoration of MSCShh+VEGF-D siRNA group was reduced.Conclusion:Shh gene can promote BMSCs differentiation to endothelial cell through Shh/VEGF-D signal axis. |
