| Objective:In recent years,stem cell transplantation therapy is a potential therapeutic method for ischemic stroke.Endothelial progenitor cells(EPC)are also one of the stem cells.Endothelial progenitor cells are pluripotent cells that can differentiate into endothelial cells in bone marrow,peripheral blood,or umbilical cord blood.Under specific conditions,they can be induced to differentiate into vascular endothelial cells and have the effects of neovascularization and endothelium.However,animal experimental studies have shown that transplanted endothelial progenitor cells are difficult to survive in areas of post-stroke inflammation and in the absence of blood vessels,causing many difficulties in clinical applications.Therefore,exploring how the endothelial progenitor cells migrate to the ischemic region and how to orientate the infarct region to form a lumen-like structure and fuse with the original blood vessel to form a blood vessel network may provide new ideas for finding more effective treatment methods.After stroke,many cytokines,adhesion factors,etc.are released in the injured area,of which CXCL12 and VEGF are the more important ones.Studies have shown that CXCL12 can mobilize and recruit endothelial progenitor cells to participate in angiogenesis,and VEGF is an important factor involved in this process.Therefore,we hypothesize that there is a synergistic effect between CXC12 and VEGF on the proliferation,migration,and angiogenesis of endothelial progenitor cells.In the future,explore the role of both in the directional regeneration of neovascularization in the ischemic stroke ischemic area and the repair of nerve function.Methods:1.The mononuclear cells in cord blood were isolated by density gradient centrifugation,seeded in culture flasks coated with human fibronectin(FN),and cultured in specialized media for endothelial cells.The next day,after the cells were attached to the wall,the liquid was changed completely to remove the non-adherent cells,and after that,the cell was semi-fluidized according to the cell growth condition.A week or so,cobblestone-like cells grew out of the cell community and acquired EPC.Immunofluorescence was used to identify its surface antigen.2.In vitro proliferation experiments:To compare the effects of CXCL12,VEGF,CXCL12 and VEGF groups on the proliferation of EPC,CCK8 reagent(CXCL12:10 ng/ml,50 ng/m,l00 ng/ml;VEGF:10 ng/ml,50 ng/ml,100 ng/ml;three CXCL12 combined VEGF concentrations:10 ng/ml CXCL12+10 ng/ml VEGF;50 ng/ml CXCL12+50 Ng/ml VEGF;100 ng/ml CXCL12+100 ng/ml VEGF)was added after treatment with different groups of cytokines.Two hours later,the OD value at 450nm of each group was detected with a microplate reader.3.In vitro cell migration and polarization experiments:Make strips in advance using the PDMS strip mold,add different concentrations of CXCL12 and VEGF in the Petri dishes,。After spread 1×10~4 cell suspensions,the dish was incubate for20 minutes,and fixed.Actin staining was used to observe the distribution of cell actin.Transwell system coated with FN and PDL was used.After plating the same number of cells,different concentrations of CXCL12 and VEGF were added.After 24 hours,DAPI staining was performed.The number of cells that migrated to the lower layers in each group was compared using the software.3.In vitro lumen formation assay:In order to compare and analyze the control group,CXCL12,VEGF,and CXCL12 in combination with VEGF group affected and differentiated the in vitro angiogenic ability of endothelial progenitor cells.50 uL Matrigel was plated in a 96-well plate at 37°C.After incubation for 30 minutes,the digested primary 1×10~4 endothelial progenitor cells were then resuspended in ECM basal media mixed with 100μL each,and then added to a96-well plate,followed by addition of different groups of cytokines(blank control group,10 ng/ml VEGF,10 ng/ml CXCL12+10 ng/ml VEGF,50 ng/ml CXCL12+10ng/ml VEGF,100 ng/ml CXCL12+10 ng/ml VEGF)After 4 h incubation,the capillary lumen was observed under an inverted phase contrast microscope.The formation of the sample-like structure was photographed and analyzed using WimTube software and obtained the number and length of the lumen-like structures generated.SPSS 21.0statistical software was used to analyze and compare the differences between each group.Results:In the proliferation experiments,VEGF,CXCL12 compared with VEGF promoted EPC proliferate in a concentration-dependent manner;and the value reached a peak at 50 ng/ml concentration,but this trend is not continue to increase,but with increasing concentrations(100ng/ml CXCL12+100ng/ml VEGF),the proliferative capacity of EPC instead decreased,but its proliferation ability did not exceed the use of VEGF group alone,so CXCL12 combined with VEGF on the promotion of EPC proliferation capacity has the best combination of concentrations.In the migration and lumen experiments,CXCL12 combined with VEGF group was able to promote the migration and lumen formation of EPCs in a concentration-dependent manner compared with CXCL12 and VEGF alone.In the cell polarization experiment,actin in the CXCL12 group and CXCL12 combined with the VEGF group was polarized toward the band,but there was no significant change in the VEGF group.Conclusion:In the EPC proliferation,migration and tube formation,the effect of the combination of CXCL12 and VEGF cytokines within a certain concentration range is greater than a single,indicating that CXCL12 and VEGF have a combined effect,and the optimal concentration ratio,It may provide a better living environment for subsequent EPC transplantation experiments. |
