Construction Of Tissue-engineered Vascular Sinoatrial Node In Vitro

Objective To explore the methods and ways of inducing rabbit primary bone marrowderived mesenchymal stem cells(BMSCs)to differentiate into vascular endothelial cells(VECs)and pacemaker-like cells in vitro,and to construct three-dimensional vascularized tissue-engineered sinoatrial node in vitro,so to provide therapeutic strategies for biological pacing of cardiovascular diseases.Methods 1 Extraction of rabbit bone marrow mesenchymal stem cells: Bone marrow marrow was extracted and bone marrow mesenchymal stem cells were isolated and purified by whole bone marrow adherence method,and their morphology and growth and proliferation were observed.2 Induction into pacing-like cells: isolation of rabbit cardiac sinoatrial node,and separation and purification of sinoatrial node cells by differential adherence method combined with 5-bromodeoxyuridine(5-Brd U)method,observe and record growth;The sinoatrial node cell lysate was prepared by repeated freeze-thaw method,and the bone marrow mesenchymal stem cells were induced into pacing-like cells as an inducer,and the induced hyperpolarization of the surface cells of the pacemaker-like cells was detected by immunofluorescence staining.Expression of activated cyclic nucleotide-gated cation channel HCN2 and immunocytochemical staining were used to detect the expression of the pacing-like cell surface marker troponin T(c Tn T)after induction,using uninduced BMSCs as Negative control group;and the bone marrow mesenchymal stem cells were induced to induce pacing-like cell growth curve.3 Induction into vascular endothelial cells: extraction of rabbit bone marrow,isolation and purification of bone marrow mesenchymal stem cells,induction of bone marrow mesenchymal stem cells into endothelial cells by endothelial cell culture medium,observation of morphological changes under microscope and detection of cell proliferation;use of immune cells The induced vascular endothelial cell cytokines CD31 and CD34 were detected by chemical staining,and uninduced bone marrow mesenchymal stem cells were used as negative control group.The growth curve of vascular endothelial cells induced by bone marrow mesenchymal stem cells was drawn.4In vitro construction of three-dimensional vascularized tissue engineering sinoatrial node:nucleus of vascular endothelial cells,cytoplasm and pacing-like cells induced by CM-dil and DAPI fluorescent labeling,according to different groups(group A is vascular endothelial cells): pacemaker-like cells are 1:2;group B is vascular endothelial cells:pacing-like cells are 1:1;group C is vascular endothelial cells: pacemaker-like cells are2:1)will induce vascular endothelial cells and The pacing-like cells were cultured for 48 h with the scaffold Matrigel Matrigel.The "angiogenic phenomenon" in the complex was observed under an inverted microscope and statistical analysis was performed.The vascular endothelial cells and the pacemaker-like cells were 1:1.The proportions were constructed in vitro on Matrigel Matrigel,and the complexes were frozen and sectioned for three-dimensional imaging and morphological observation.Results 1 Bone marrow mesenchymal stem cells can be obtained by whole bone marrow adherence method.2 Pacemaker-like cells and vascular endothelial cells were obtained from bone marrow mesenchymal stem cells induced by sinoatrial node cell lysate and endothelial cell culture medium respectively.The surface markers HCN2,c Tn T,CD31 and CD34 were all positive by immunofluorescence staining and immunocytochemical staining.3 Three-dimensional vascularized tissue-engineered sinoatrial node can be constructed in vitro by co-culture of vascular endothelial cells and pacemaker-like cells-Matrigel matrix gel.Conclusions Bone marrow mesenchymal stem cells can be induced into pacemakerlike cells and endothelial cells by sinoatrial node cell lysate and endothelial cell culture medium.Three-dimensional vascularized tissue-engineered sinoatrial node in vitro can be constructed by using Matrigel matrix glue as scaffold material.Figure 47;Table 4;Reference 105...