Jagged-1-mediated Activation Of Notch1 Signaling Promotes Tissue Engineered Valve Recellularization

Objective: To study the role of Notch1 signaling in the construction of tissue-engineered heart valves(TEHV),and then further explore the mechanism by culturing cells.Methods: 1.The endothelial cell induction medium containing VEGF and FGF was used to induce the differentiation of ADSCs.The ability of ADSCs to differentiate into endothelial cells was examined by observation of cell morphology,protein immunoblotting assay to detect the expression of v WF and tube formation assay;2.Preparation of decellularized valve scaffolds using enzymatic digestion in combination with Triton X-100.Identification of decellularization effect by pathological staining,scanning electron microscopy,etc.;3.The decellularized valve scaffold was coated with Matrigel,and the ADSCs-induced cell population was used as seed cells to inoculate the valve scaffold for compound culture.Notch signaling pathway activators and inhibitors(jagged-1 and dapt),the valve after compound culture was identified by HE staining,scanning electron microscope,and DNA content determination;4.The seed cells were cultured with endothelial cell medium containing jagged-1 protein and dapt respectively,and the cells were proliferated by cell proliferation experiments and flow cytometry.To study the effect of Notch signaling pathway on the growth cycle of seed cells,cell invasion and scratch experiments to study the effect of cell migration ability,Western blotting and cellular immunofluorescence were used to detect EMT-related proteins(E-Cad,N-Cad,SMA))and the expression of adhesion-related proteins(FN,Paxillin).Changes in cell adhesion were further investigated by cell adhesion experiments.Results: 1.The expression of v WF can be detected after induction of ADSCs,and the expression of v WF increases with the increase of induction days,and it has a certain ability to form tubes after induction.2.The HE staining of the valve stent prepared by the enzymatic digestion method and Triton X-100 method showed no obvious cell nuclei.Scanning electron microscope and Masson staining showed that the extracellular matrix was basically preserved;HE staining of the recellularized valve showed that there were discontinuous cells on the surface of the valve.There were no obvious cellular components in the valve interstitium,and the cells were closely connected with the extracellular matrix under the electron microscope.3.After activating Notch signal,the proliferation ability of seed cells was enhanced,N-Cad,SMA,FN,Paxillin were increased,the expression of E-Cad was decreased,and the surface cells of compound cultured valve were more covered.Conclusion: 1.ADSCs can differentiate into endothelial cells under the induction of VEGF and FGF,and can be used as seed cells.2.The activation of Notch1 signaling pathway by Jagged-1 can promote the proliferation,migration and adhesion of seed cells.3.Activation of Notch1 signaling pathway can promote TEHV endothelialization.