| Objective: To induce the umbilical cord blood-derived CD34~+ cells differentiate into endothelial cells(ECs) in vitro,and investigate the activation pattern of signal transducers and activators of transcription(STAT)induced by vascular endothelial growth factor(VEGF) in the process of the differentiation,gain an insight into the molecular mechanism and signal transduction pathway of the differentiation of CD34~+ cells into ECs.Methods: CD34~+ mononuclear cells were selected by magnetic beads coated with antibody CD34.Cells were transferred onto regenerated silk fibroin film(SF) and gelatin coated dishes, and cultured in M-199 medium containing vascular endothelium growth factors(VEGF) and basic fibroblast growth factors(bFGF), morphology of the cells was observed and cell surface marker CD34,CD31 and vWF was assessed by fluorescence activated cell sorter(FACS)analysis,the endothelial-specific Weibel-Palade body was also observed by electronic microscope,then identified the adherence and proportion of the CD34~+ cells-derived ECs under these two substrates.WI-38 cell cultured on SF were infected with the recombinant adenoviruses Ad-VEGF165-PolyA-promoter-Ang-1,and the expressions of VEGF165 and Ang-1 were detected by a fluorescence microscope,RT-PCR and ELISA.Then the SF coated with genetically modified cells was transplanted onto the medium of CD34~+ cells, to compare proportion of CD34~+ cells-derived ECs with the inducing way of cytokine by observing cell morphology and fluorescence activated cell sorter(FACS)analysis.Moreover,total cellular JAK2 and STAT5 and tyrosine phosphorylated protein stimulated by VEGF at different time points(0d,7d,and 14d) during ECs differentiation were detected by Western blot;CD34~+ cells were stimulated by VEGF(50ng/m1)for different time(5,10,30,60min)to detect the tyrosine phosphorylation and nuclear translocation of STAT-3 by Western blot and immunocytochemistry methods;ATWLPPR,an effective peptide screened from phage epitope library by affinity for membrane-expressed VEGFR2 and blocking the binding of VEGF to VEGFR2,was used to determine whether the activation of STAT3 pathway induced by VEGF was blocked;AG490,an specific inhibitor of JAK-STAT signal transduction pathway ,was added to the 14d cell to determine the tyrosine phosphorylation of STAT3 by Western blot, at the same time, detected the condition of CD34~+ cell growth and differentiation cultured with AG490 by observing cell morphology and FACS analysis.Results: (1) The number of CD34~+ cells reached to the highest level at the 14th day;In FACS assay,more than 70% of cells were positive-stained for CD31 after 10 days of differentiation,the expression of vWF in the cells were approximately 80% after 14 days of differentiation;The endothelial-specific Weibel-Palade body were detected in the cytoplasm by electronic microscope; The average adhesive rate were approximately above 80%,and the differentiate rate were approximately above 60%. SF had no negatively influence to the differentiation of CD34~+ cells.(2) WI-38 cell grew well on SF, and after being infected with Ad-VEGF165-PolyA-promoter-Ang-1 they obtained strong green fluorescence. Moreouver, ectogenic VEGF165 and Ang-1 genes could transcribe in WI-38 cell,the cell factor Ang-1,VEGF165 and FGF2 could expressed unremitly by ELISA. The SF coated with genetically modified cells was co-culture with CD34~+ cells for 7 days,then cell multiplication and differentiation rate,expression of CD31 were all higher than other groups.The SF coated with genetically modified cells could induce CD34~+ cells differentiate into ECs better.(3) The amount of JAK2 protein was similar at 0,7,and 14d without VEGF stimulation,with the differentiation of cells into ECs, the amount of tyrosine phospho-JAK2 induced by VEGF increased markedly;Both total cellular and tyrosine phospho-STAT3 expression increased markedly during ECs differentiation;VEGF stimulation of 14d cell resulted in a rapid and transient tyrosine phosphorylation and nuclear translocation of STAT-3, VEGF stimulation to the expression of tyrosine phospho-STAT3 had time dependence;The phosphorylation of STAT-3 failed to be activated by the co-culture with ATWLPPR and VEGF; The phosphorylation of STAT-3 still could be activated by the co-culture with AG490 and VEGF, furthermore the co-culture CD34~+ cells could partially differentiate into ECs.These showed that STAT signaling pathway via VEGFR2 may take part in the signal mechanism of ECs differentiation from CD34~+ hematopoietic stem cells stimulated by VEGF, at the same time other signaling pathway could participated the ECs differentiation.Conclusion: (1)Human umbilical cord blood-derived CD34~+ cells were successfully induced into ECs, and silk fibroin materials can be used as scaffolds; (2)The SF coated with genetically modified cells could induce CD34~+ cells differentiate into ECs better; (3)STAT signaling pathway via VEGFR2 may take part in the signal mechanism of ECs differentiation from CD34~+ hematopoietic stem cells stimulated by VEGF.So we can get tissue engineering prosthesis with the ability of forming capillary network in the shortest time to maintain the living of parenchymal cells. Key words: CD34~+ hematopoietic stem cells; regenerated silk fibroin film(SF);... |
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