| Background and objective: Bone defect disease is a kind of disease that seriously affects people's quality of life and even threatens life.The regeneration of blood vessels and the reconstruction of blood circulation are of great significance in orthopedics including the healing of bone defects and fractures,lower limb blood reconstruction and healing in diabetic foot patients,and the prevention and treatment of avascular necrosis of femoral head.Vascular regeneration is the key to such diseases.The development of regenerative medicine and tissue engineering provides new methods and ideas for the treatment and rehabilitation of such diseases.One of the important ideas to promote angiogenesis is to select the seed cells for transplantation.Bone mesenchymal stem cells(BMSCs)can be one of the candidates for seed cells.At present,the research on the differentiation of mesenchymal stem cells into endothelial cells includes induction by cytokines and cell co-culture methods.In this research,cell co-culture methods are used for induction.The heat shock method is a kind of cell treatment method in tissue engineering,and has made important progress in the field of stem cell differentiation.It has not been reported that heat shock treated endothelial cells can induce mesenchymal stem cells to differentiate into endothelial cells.The differentiation of bone marrow mesenchymal stem cells into endothelial-like cells is regulated by a variety of factors.Notch signaling pathway plays one of important roles in the process of cell differentiation and neovascularization in ischemic disease.This research mainly includes the observation and exploration of the ability and feasibility of heat shock-treated human umbilical vein endothelial cells(Human Umbilical Vein Endothelial Cells,HUVEC)inducing bone marrow mesenchymal stem cells into vascular endothelial cells;and includes the discussion of the ability of induced bone marrow mesenchyme stem cells forming blood vessels;and study the role of Notch signaling in this differentiation process.Methods: 1.Human bone marrow-derived mesenchymal stem cells were extracted and cultured,and cells were cultured to the third generation(P3).Thirdgeneration(P3)to seventh-generation(P7)cells was selected for research.And observe the cells under the microscope.Flow cytometry was used to identify the expression on cell surface of CD90,CD105,CD34,CD14,CD31,CD45,CD144,VEGFR2,v WF.2.Verified the differentiation ability of extracted bone marrow mesenchymal stem cells.Osteogenic induction medium and adipogenic induction medium were used respectively for osteogenic and adipogenic differentiation,which demonstrates the ability of osteogenic and adipogenic differentiation of mesenchymal stem cells.Osteogenic differentiation ability were measured with alizarin red staining and cell alkaline phosphatase activity assay,and adipogenic differentiation ability was verified with oil red O staining.3.Human umbilical vein endothelial cells were extracted and cultured,and the second-generation(P2)to sixth-generation(P6)cells were used for experiment.Observed the morphology of the cells under an inverted microscope.Detected the expression of CD105,CD144,CD31,CD34,VEGFR2,v WF,CD14,CD45 with flow cytometry.4.Performed heat shock treatment on human umbilical vein endothelial cells,and then collected the human umbilical vein endothelial cells digested from the culture flask in a centrifuge tube.Placed the tube in a water bath at 40 ? 42 ? for1 hour.Umbilical vein endothelial cells and bone marrow-derived mesenchymal stem cells were co-cultured,and the cells were induced with non-contact cocultivation in the Transwell culture plate.Placed in a cell culture incubator for 7and 14 days.After 7 days and 14 days of culture,the cells were collected and cellular immunophenotypic antigens CD31,CD144,VEGFR2,and v WF were detected by flow cytometry.Preliminary verification of differentiation results was performed.5.Uninduced cells,cells after 7 days of induction and cells after 14 days of induction were collected respectively,and cellular immunofluorescence was performed for detection of CD31,CD144,VEGFR2,v WF,UEA-1.Observed under a fluorescent microscope,and the optical density value(IOD value)was analyzed by computer software.6.Undifferentiated cells were collected as a control group,and cells after 7days and 14 days of induction were collected respectively.Cells were inoculated on Matrigel.The inoculated cells are placed in a incubator for 24 hours.Then observed under an inverted phase contrast microscope,and the length of blood vessels under the microscope was calculated and statistically analyzed with computer software to analyze the ability of angiogenesis in vitro.7.Cells 7 days and 14 days of induction and uninduced human bone marrow mesenchymal stem cells(control group)were collected.They were resuspended in Matrigel respectively.Calculated and adjusted the cell density of each group to ensure the same cell density in each group.Same volume of resuspended Matrigel was injected into the back of nude mice subcutaneously for allogeneic transplantation.After 14 days,the graft was taken out for fixation,embedded in paraffin,and tissue sections were taken for hematoxylin-eosin staining(HE staining).And immunochemistry was conducted to detect CD31,CD144,VEGFR2,v WF.Observed under a pathological microscope to analyze the ability of angiogenesis in vivo.8.Uninduced stem cells were collected as a control group,and cells induced for 7 and 14 days were collected as an experimental group.Protein and m RNA was extracted.Real-time quantitative PCR was conducted to detect the expression of Notch1,Dll4 and Hes1,and Western Blot was performed to analyze the expression of Notch1,Dll4 and Hes1 protein.9.Add the Notch pathway inhibitor FLI-06 in the process of cell differentiation to explore the regulatory role of Notch signaling pathway.Uninduced stem cells were collected as a control group,and cells induced for 7and 14 days were collected as an experimental group.Cells were inoculated on Matrigel to repeat the angiogenesis experiment in vitro.Observed under an inverted phase contrast microscope,and length of blood vessels were calculated with computer software to analyze the ability of blood vessels formation in vitro.Results: 1.Fibroblast-like aggregation and growth can be observed at 4th day of induction.Cells grow rapidly and form large colonies in 5-10 days.Some colonies are arranged in a swirling shape,and the colonies gradually merge together.After passage,the growth speed is accelerated,and the cells grow into single shape.The cells arrange in regular direction,and gradually become spindleshaped.Identification of cell surface markers by flow cytometry showed that human bone marrow mesenchymal stem cells expressed CD90,CD105 and CD34,and underexpressed CD14,CD31,CD45,CD144,VEGFR2,and v WF.CD34 and CD45 act as hematopoietic stem cell markers,and the expression level on bone marrow mesenchymal stem cells is low,still within acceptable range.2.Bone marrow mesenchymal stem cells have obvious morphological changes after 7 days of osteogenic induction,with a paving stone-like appearance and calcium nodule formation.After staining with alizarin red,the calcium salt is stained red,and it is more obvious on the 7th day.On the 14 th day,a larger number and more calcium nodules can be seen.As the induction time increases,the alkaline phosphatase activity increases.Compared with uninduced bone marrow mesenchymal stem cells,there is a significant increase in alkaline phosphatase at7 and 14 days of induction,and the difference was statistically significant(p<0.01).Around 7 days after induction,many translucent and dense lipid droplets were formed in the cells.As the induction time increases,the intracellular lipid droplets gradually increased.It has been verified that the differentiation ability of mesenchymal stem cells is satisfactory,and can be used as seed cells for cell differentiation.3.HUVEC adheres to the flask wall 24 hours after cell inoculation,with an oval,paving stone-like morphology,rich encapsulation,centered elliptical nuclei,and clear boundaries.Flow cytometry shows high expression of CD105,CD144,high expression of endothelial cell surface marker CD31,and moderate expression of VEGFR,v WF.Expression of CD34 on endothelial progenitor cells and hematopoietic stem cells is low,and low expression of CD14 and CD45.4.After 7 days and 14 days of co-culture induction,the morphology of human bone marrow mesenchymal stem cells changed,losing the elongated spindle and swirling shapes,close to paving stone-like changes.Flow cytometry showed that,except for the increase of CD31 expression,the expression of CD144,VEGFR2 and v WF changed significantly,among which the expression of CD144 increased most significantly.5.Further analysis of the surface antigen expression after cell differentiation by cell immunofluorescence and fluorescence intensity analysis showed that the expression of vascular endothelial-specific antigens CD31 and CD144 after 7 and14 days of induction was significantly higher than that of the control group(P<0.05).VEGFR2 and the expression of v WF was higher than that of the control group(P <0.05),which was consistent with the results of flow cytometry;UEA-1 was also expressed in endothelial cells,and its expression was also higher than that of the control group(P <0.05).6.Under the inverted microscope,it can be seen that the uninduced cells are scattered or aggregated,and there is no network structure formation.The cells induced for 7 days can be seen a small number of lines formed,and the structure increased in the 14 days.But compared with endothelial cells,there are no net structure.Endothelial cells in Matrigel can quickly form a large number of lines and polygonal structures in only 24 hours,forming a network structure.7.Undifferentiated mesenchymal stem cells cannot form a tubular structure in vivo,and the cells are scattered randomly.Cells induced for 7 days can form a regular directional line structure within 14 days in vivo,and have a tendency to form a tubular structure.The 14-day induced cells was similar to that of the 7-day induced group,with a certain direction,and the formation of a stripe structure.Histochemical results showed that except for the small amount of CD144 expressed in the control group,CD31,VEGFR2 and v WF were hardly expressed in the control group.The expression of CD144 increased in the 7-day induction group and the 14-day induction group.CD31,VEGFR2 and v WF began to express in the 7-day induction group and the 14-day induction group,and showed an increase in expression as the induction time increased.8.Western Blotting and q PCR results showed that the expression of Notch1 increased in the 7-day induction group and the 14-day induction group compared with the control group,the difference was statistically significant(p <0.05).The expression level of Dll4 was slightly increased after 7 days of induction compared with the control group.After 14 days of induction,the expression of Dll4 increased significantly,and the difference was statistically significant(p <0.05).The expression of Hes1 increased significantly at 7 days after induction,but there was no significant difference of Hes1 expression between 14-day induction group and 7-day induction group.9.In the angiogenesis experiment in vitro,after adding the inhibitor,the tube structure and the line structure could not be formed with the cells after 7 days and14 days of induction.The ability to form a network structure was significantly weakened.Compared the control group with 7-day induction group and 14-day induction group,the difference was no longer significant,and the difference was not statistically significant(P> 0.05).Conclusion: Heat shock treated human umbilical vein endothelial cells can make bone marrow mesenchymal stem cells to differentiate into endothelial cells to a certain extent,and the induced cells have certain vascular forming ability and tendency.The Notch signaling pathway regulates the differentiation of bone marrow mesenchymal stem cells into endothelial cells.Blocking the Notch signaling pathway can inhibit the differentiation of mesenchymal stem cells into endothelial cells,and can inhibit their angiogenic capacity in vitro. |
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