The Study Of BMSCs Differentiation Into Endothelial Cells And The Role Of Notch Signal Pathway In It

Backgroud:With the development of modern society,coronary atherosclerotic heart disease?CHD?morbidity and mortality is obviously higher than ever before and the acute myocardial infarction and cardiac insufficiency is an important cause of death.Due to the feature of non-renewable,after myocardial infarction,infracted myocardium is replaced by fibrous tissue dand hyperplasia scar tissue.Infarction areas lost contraction and the conduction ability of normal myocardium,results in myocardial remodeling and cardiac insufficiency.Adjacent myocardium with infarction area will become hibernating myocardium due to ischemia and hypoxia.Traditional treatments about myocardial infarction include drug therapy,interventional therapy and surgical coronary artery bypass graft surgery.They have saved ischemic myocardium and improved the prognosis of patients with myocardial infarction to a certain extent.But infarcted myocardial cell cannot acquire rebirth throuth these methods.Especially in patients with diffuse vascular lesions and small vascular lesions,intervention and surgical treatment The effect of intervention and surgical treatment is poorer,and long-term oral antiplatelet drugs after stenting is needed,the bridge vessels after bypass surgical may come to atherosclerotic occlusion even again.In recent years the development of tissue engineering and regenerative medicine provides a new hope for the treatment of myocardial infarction?MI?.Promoting angiogenesis through cell transplantation therapy will save ischemic myocardium,hibernating myocardium and figure out a new direction for MI treatment,This treatment is also called "therapeutic angiogenesis".Many kinds of cells have been studied during the clinical and basic research about cell transplantation,such as Umbilical vein endothelial cells,endothelial progenitor cells,embryonic stem cells,mesenchymal pluripotent stem cells.The bone marrow mesenchymal stem cells?BMSCs?is a group of cells acquired from bone marrow,unlike the hematopoietic stem cells,has the ability of self-renewal and differentiation potential towards other cells under right conditions in vitro.they can differentiate into multiple cell types,be suitable for autologous transplantation,easy to import exogenous genes,thus makes them popular candidate seed cells.The therapeutic angiogenesis role after transplantation which BMSCs plays mainly attributed to their differentiation into endothelial cells,cardiomyocytes,paracrine effect to promote angiogenesis and immune regulation against inflammatory injury after infarction,etc.BMSCs to endothelial cells differentiation are regulated by various factors,the mechanism underlying is still in research.Notch signaling pathway exists widely in species,many cell types in a highly conservative way.It mainly work through contact conduction between cells and play an important role in deciding cell differentiation and destiny.Notch signaling is indispensable in the normal development of the cardiovascular system,the abnormal activation or lack of it can lead to cardiovascular anomalies even death.Notch signaling also plays a key role in the angiogenesis induced by ischemia,endothelial cell differentiation and arteriovenous selection.This study focused on the feasibility of differentiation from human BMSCs to endothelial cells in vitro and the role which Notch signaling pathway plays in it,so as to provide experimental basis for improvement and development of BMSCs transplantation in ischemic heart disease.Part1 human BMSCs culture and induced differentiation into endothelial cells in vitroObjiective:To culture human BMSCs in vitro,and try to induce them into endothelial cells,to observe the cell morphology and the ability into of blood vessels formation,changes of cell surface markers,confirm the endothelial differentiation potential of BMSCs.Methods:?1?we cultured primary human BMSCs to the third generation for use,then tried to induce the BMSCs into endothelial cells in the presence of VEGFA165 and b FGF.Cells morphological changes were observed under microscope before and after induction.?2?The expression of endothelial cell surface markers Flk-1 and vWF were detected by immunohistochemical metheds during different time point:study objiect were divided into two group as control group and induced group.Immunohistochemical detection were played in day 5,day7,day10.Cells before and after inducing were cultured in matrigel to observe formation ability.Ac-LDL uptake ability was detected too.?3?investigate the expression of Notch ligand DLL4 on BMSCs before and after inducing by immunohistochemical metheds.try to confirm that DLL4 is invoved in the differentiation processResult:?1?human BMSCs have the morphological feature of Spindle and fusiform under microscope.After the inducing of VEGFA and b FGF,Cells morphology changed into flat,Short spindle.?2?After the inducing of VEGFA165 and b FGF,Immunohistochemical evaluation showed that cells express Flk-1 and vWF endothelial-specific markers.The induced cells were cutured in the matrigel and showed greater ability of forming capillary struture.The ablity of swallowing ac-LDL were observed too.?3?After the differentiation form BMSCs to endothelial cells,significantly rise of DLL4 protein expression is observed?P<0.01?.Concusion:?1?The combination use of VEGF and b FGF for differentiation of h BMSCs to endothelial cells can work,the best inducing concentration is.The differentiated cells are similar with endothelial cells in morphology and function.?2?During the differentiation,Cells express more endothelial cell marker and the ability of capillary forming and ac-LDL uptakeing are expressed.?3?Protein expression of DLL4 elevated significantly afer the differentiation?P<0.01?.Part2 The construction and transfection of DLL4 interference plasmid and overexpression LentivirusObjiective:To construct DLL4 interference and overexpression cell models by genetic vector technology and transfect them into hBMSC.To make foundation for the future study of Notch signal pathway in endothelial differentiation.Methods:?1?The target gene and vector were cleaved by double digestion,then their PCR produce were connected and transfected into competent cells.Positive colonies were screened and the plasmid carrying target DLL4 interference sequence were extracted.?2?The two segments of DLL4 gene were acquired by oligo mix technology,and recombinant were completed in LV5.Positive colonies were screened in competent cells.DLL4 gene were transfected into h BMSCs after Packaging and purification.?3?The effection of interference and overexpression cell models Was verified by WB test.Results: The DLL4 interference and overexpression cell models changed the expression of DLL4 protein as expected.Conclusion: The DLL4 interference and overexpression cell models were effective and can be used in next experiment.Part3 The effect of Notch signal pathway on the differentiation from hBMSCs to endothelial cellsObjiective:To confirm the changes of Notch signal pathway during the differentiation,BMSCs were transfected by interference plasmid and overexpression lentivirus to rise and reduce the DLL4 expression.We aim to investigate the effect of Notch signal changes on differentiated cells characteristics so as to apply it in clinical use,promote the proliferation and angiogenesis ability of differentiated cells.Methods:?1?BMSCs transfected were induced by VEGF and bFGF into endothelial cells.The expression of endothelial cell surface markers Flk-1 and vWF were detected by immunohistochemical metheds and WB.?2?The downstream molecules of Notch signaling pathways such as Notch intracellular fragments?NICD?,HES1,HES5 expression were detected by WB.BMSCs were also disposed by Notch1 specific antagonist DAPT and DLL4 fragments to investigate endothelial cell marker expression level,capillary forming ability and Ac-LDL swallowing ability.Results:?1?Weak positive Flk-1and v WF can be detected in h BMSC with DLL4 interference,moderate positive Flk-1and vWF can be detected in hBMSC with overexpressed DLL4.?2?HBMSC with overexpressed DLL4 expressed more NICD?Hes1?Hes5?Flk-1?v WF than hBMSC group,hBMSC with DLL4 interference express less endothelial marker and Notch downstream.?3?HBMSC with overexpressed DLL4 had stronger capillary forming ability.hBMSC with DLL4 interference had counter-productive results.DAPT blocking Notch in the late phase of differentiation results in stronger capillary forming ability in vitro.Conclusion:DLL4-Notch pathway may act as promoter in the h BMSC differentiation into endothelial cells induced by VEGF and b FGF,Notch blocking in the late phase of differentiation results in stronger capillary forming ability in vitro.Part4 Carnosol promotes endothelial differentiation under oxidative stressObjiective:To investigate the anti-oxidative effect of Carnosol and positive effection on endothelial differentiation of MSCs,make an foundation for combining the antioxidants and Notch signal in endothelial differentiation.Methods:?1?We cultured the rat MAPCs and detected the impact of H₂O₂ and Carnosol on cell proliferation and viability.?2?Rat MAPCs were induced by VEGF,The group pretreated by Carnosol and control group were treated by H₂O₂,ROS and Caspase-3 activity were detected.?3?The endothelial makers and Nrf-2 were detected after 10 days inducing.Results:?1?H₂O₂ downregulated the rat MAPC proliferation and viability.IC50 is 27um/L.The Carnosol precondition can protect the cell,the best protecting concentration is 0.2um/L.?2?H₂O₂ upregulated the ROS level,Caspase-3 activity and cell apoptosis.Carnosol precondition can counteract this effect.?3?OCT-4,Flk-1,CD 31 of H₂O₂ pretreated group were lower than control.but Carnosol precondition group expressed more endothelial marker and Nrf-2 than H₂O₂ group.Conclusion:H₂O₂ upregulated the ROS level,cell apoptosis and downregulated endothelial marker expression of rat MAPCs during endothelial differentiation,and Carnosol precondition can counteract this effection.