Resarch Of Notch Signaling Pathway Regulating The Differentiation Of Human Adipose Derived Mesenchymal Stem Cells Into Endothelial Cells

Objective:To explore the function of Notch signaling pathway in the differentiation of human adipose derived mesenchymal stem cells(hADSCs),induced human adipose derived mesenchymal stem cells to differentiate into endothelial cells by VEGF165,interposed the differentiation by y-secretase inhibitor(DAPT),gauged the changes of differentiation in cells,the expression of notch signaling,the ability of migration and angiogenesis in cells.Methods:1,Three cases of adipose tissue were derived from 3 patients in the plastic surgery of Affiliated Hospital of Zunyi Medical College,the patients had agreed and signed the informed consent,the tissues were collected under aseptic conditions in the operation room,the hADSCs were extracted from the adipose tissue by enzyme digestion and shear apparatus,centrifugation,the hADSCs were fostered and passed with fetal bovine serum DMEM culture medium,the cell morphology was observed under inverted microscope.Flow cytometry was used to detect the expression of CD29,CD31,CD34 and CD44 which was the cell-surface marker;induced the third generation cell to differentiate into endothelial cell by VEGF165.Flow cytometry was used to detect the expression of endothelial cell surface antigen in 14 days.2,Took the third generation hADSCs and divided them into two groups:? control group:hADSCs;? induction group:hADSCs+VEGF165(50ng/mL).When the cells were cultured for 7 and 14 days in each group,the mRNA expression of Notch1,Notch 14 and D114 was detected by the real time quantitative PCR,the protein expression of Notch1,Notch14 and D114 was detected by western bolt.3,Took the third generation hADSCs and divided them into three groups:?control group:hADSCs;? induction group:hADSCs+VEGF165(50ng/mL);? Intervention group:VEGF165(50ng/mL)+hADSCs+ DAPT(2.5ug/ml,lOug/ml,25ug/ml);When the cells were cultured for 7 and 14 days in each group,the expression of VE and cadherin CD31 antigen was detected by flow cytometry respectively.The ability of migration ability was detected by transwell.4,Took the third generation hADSCs and divided them into 3 groupss,the treatment and grouping were as the same as the assay of cell-migration,when the cells were cultured for 7 and 14 days in each group,transplanted 2*104 cells on the 96 hole plate which were covered with matrigel matrix gel,put them in the 37? incubator,6 hours later,photographed them by microscope to analyze the ability of angiogenesis in vitro.5,Took the third generation hADSCs and divided them into 3 groups,the treatment and grouping were as the same as the assay of cell-migration,the cells were cultured for 7 days in each group,the melted matrigel matrix gel was mixed with corresponding culture medium of each group,and then blended each mix mixture with cells of three groups individually,implanted the last mix into the nude mice in the side rib section.The mice were killed by cervical dislocation 7 days later,removed out the implanted matrigel glue,angiogenesis was observed in the gel,the specimens were sectioned for CD31 immuno staining to analyze the ability of angiogenesis in vivo.Results:1,Mesenchymal stem cells(MSCs)were extracted from human adipose tissue,their shapes were spindle shaped and stellate,the growth form of them were like radioactive colony.The cell phenotype was detected by flow cytometry,the result showed that the high expression of CD29(96.31%)and CD44(98.91%)was found in the third generation hADSCs,the expression rate of CD31 and CD34 which was the evidence of endothelial cell was negative;When the cells were induced for 14 days,the expression rate of CD31(83.38%)and CD34(92.81%)were positive,while the positive expression rate of CD29 and CD44 had reduced.2,The result of qPCR showed that the mRNA of Notch1,Notch4 and D114 were expressed in cells in the control groups and induced groups,the relative expression in the induced groups was higherer than the control groups.The relative expression ratio of the cells which were induced for 14 days was higher than 7 days,the differences were statistically significant(P<0.05);Western blot showed that the protein of Notchl,Notch4 and D114 were expressed in each group,the relative expression in induced groups was higher than the control groups,the relative expression ratio of 14 days were higher than 7 days,the differences were statistically significant(P<0.05).3,The positive rate of VE-cadherin and CD31 showed the same trend as the flow cytometry;the positive rate in control groups was the lowest,it was less than 1%;the positive rate of 14 days was higher than 7 days in the Induction groups and intervention groups;the positive rate in the induction groups was higher than the intervention groups;the positive rate in low concentration DAPT groups was higher than the high concentration DAPT groups,the differences were statistically significant(P<0.05).In the cell migration experiment,the ability of cell-migration was the strongest in the induced groups,the ability of cell-migration in the intervention groups was lower than the induced groups,the ability of cell-migration was the weakest in the control groups;the ability of cell-migration of 7 days groups were stronger than 14 days,the ability of cell-migration in low concentration DAPT groups was higher than the high concentration DAPT groups,the differences were statistically significant(P<0.05).4,The experiment of angiogenesis with matrigel in vitro,the form of cells was in separation state in the control groups,no angiogenesis;there formed some closed polygon structures and complex reticular structures in the induction groups;there were some cells ranked in lines and formed some network junctions,the ability of angiogenesis in the induced groups was stronger than the intervention groups;the ability angiogenesis in low concentration DAPT groups was stronger than the high concentration DAPT groups,the differences were statistically significant(P<0.05).5,The strength relation of angiogenesis invivo was as the same as the angiogenesis in vitro,the result of immunochemical staining showed that the inner wall of the lumen was endothelial cells,it is confirmed that the structure was vascular cavity;there were many lumen like structures in the induced groups;the number of lumen was smaller in intervention groups than the induced groups(P<0.05);in the intervention groups,the number of lumen in the low concentration DAPT groups was bigger than the high concentration DAPT groups,(P<0.05);Lumen was occasionally seen in the control groups.Concdusion:(1)VEGF can increase the expression of Notch signaling pathway in the differentiation of hADSCs into endothelial cells(2)Block of the Notch signaling pathway can inhibit the differentiation of hADSCs into endothelial cells.(3)Block of the Notch signaling pathway can inhibit the ability of cell-migration of the endothelial cells which were differentiated by hADSCs,it can also inhibit the ability of the angiogenesis in vivo and in vitro.