Conditioned Medium Derived From FGF-2-modified GMSCs Enhances Migration And Angiogenesis Of HUVECs

Background and PurposesPeriodontitis,fractures,deformities,and surgical resection of tumors can cause bone tissue defects and interfere with normal function and configuration.Therefore,it is great significance to promote the regeneration of these damaged bone tissue in clinical treatment.It has been widely recognized that mesenchymal stem cells(MSCs)offer a potential strategy for tissue repair and wound healing.However,MSCs direct transplantation has some limitaions,such as the poor engraftment,a relatively short life span and potential tumorigenesis of transplanted MSCs.And some researchers have proved that MSCs may regulate the microenvironment of the lesion site by paracrine,thereby promoting the repair and regeneration of the injured site.Therefore,conditioned medium(CM)derived from mesenchymal stem cells appears to be a promising alternative therapy for MSCs.Angiogenesis plays an important role in tissue repair and regeneration,and promoting angiogenesis can accelerate bone tissue regeneration.Conditioned medium derived from mesenchymal stem cells(MSC-CM)possesses pro-angiogenesis,but the growth factors in MSC-CM are limited in type and low concentration,which cannot meet the needs of tissue regeneration and needs to be optimized.Lentiviral transfection technology can improve the original genome sequence to change the type and quantity of factors secreted by cells.Fibroblast growth factor-2(FGF-2)has been proven to be an effective angiogenic factor.Not noly can it promote cell proliferation and maintain the stemness of mesenchymal stem cells,also it can promote the expression of other pro-angiogenesis related factors(such as VEGF-A).Thus,in this study we hope to optimize CM from human gingival mesenchymal stem cells(hGMSCs)by overexpression of the FGF-2 gene,so as to obtain optimized conditioned medium promoting blood vessel formation.Materials and Methods1.Isolation,culture and identification of human gingival mesenchymal stem cellsHuman GMSCs were isolated and cultured by using tissue block enzyme digestion method and purified by using monoclonal technology.And proliferation efficiency was evaluated by clone formation rate.Flow cytometry was used to identify surface markers of hGMSCs,and multidirectional differentiation potential of hGMSCs were determined by osteogenic staining and adipogenic staining.2.Construction and identification of LV-FGF-2+-hGMSCsLentiviral transfection technology was used to construct hGMSCs overexpressing FGF-2 gene(LV-FGF-2+-hGMSCs)and the control cells(LV-vector+-hGMSCs).The expression of green fluorescent protein was observed by using fluorescence microscope,and qRT-PCR technology was used to detect the expression of FGF-2 gene in hGMSCs.3.Acquisition of LV-FGF-2+-hGMSC-CM and detection of angiogenesis-related factors(FGF-2?VEGF-A?TGF-?)LV-FGF-2+-hMSC-CM were cultured by serum-free DMEM medium,the supernatant was collected,and concentrated by using 10KDa ultrafiltration centrifuge tube to obtain the CM(LV-FGF-2+-hGMSC-CM).Elisa technology was used to detect the concentrations of FGF-2,VEGF-A and TGF-? in LV-FGF-2+-hGMSC CM.4.Detection of the effect of LV-FGF-2+-hGMSC-CM on human umbilical vein endothelial cells(HUVECs)angiogenesis and migration in vitro.(1)qRT-PCR and westernblot were used to detect the gene and protein expression of angiogenesis-related markers(PLGF,SCF,VEGFR2)in HUVECs.Cellular immunofluorescence technology was used to detect the expression of VEGFR2.(2)The effect of LV-FGF-2+-hGMSC-CM on HUVECs angiogenesis was determined by tube experiment in vitro.(3)Transwell chamber was used for cell migration experiment to detect the effect of LV-FGF-2+-hGMSC-CM on HUVECs migration5.Detection of the ability of LV-FGF-2+-hGMSC-CM promoting HUVECs angiogenesis in mice.The matrigel were mixed with HUVECs,HUVECs and hGMSC-CM or HUVECs and LV-FGF-2+-hGMSC-CM,and then injected subcutaneously into the mice(matrigel was regarded as coontrol).After 7 days,the materials were taken out,and the ability of angiogenesis was identified by improved CD31 immunohistochemistry and analyzed by statistics.Results1.Crystal violet staining results showed that the clone formation rate of hGMSCs was 42.7%.Flow cytometry results showed that hGMSCs highly expressed CD44(100%),CD90(99.9%)and CD 105(99.4%),which are markers on the surface of stem cell,and low expressed CD34(5.8%)and CD45(0.7%)which are markers on the surface of hematopoietic stem cell.After 21 days of osteogenesis induction,hGMSCs formed obvious mineralized nodules,and after 21 days of adipogenesis induction,hGMSCs formed obvious red lipid droplets.2.Stronger green fluorescence in hGMSCs infected by lentivirus were observed under fluorescence microscope when MOI=40.The qRT-PCR results showed that the FGF-2 gene expression in the experimental group was significantly higher than that in the empty vector group and the normal hGMSCs group.3.After supernatant was concentrated about 50 times by using 10 kDa ultrafiltration centrifuge tube,CM was derived.Elisa assay showed that,compared with the empty vector group and the normal hGMSC group,the conditioned medium derived from the experimental group contained more FGF-2,VEGF-A and TGF-?.4.(1)The qRT-PCR and western blot assay results showed that the gene and the protein expression of angiogenesis-related markers(PLGF,SCF,VEGFR2)in LV-FGF-2+-hGMSC-CM group were significantly higher than that in the ECM group,hGMSC-CM group,and LV-vector+-hGMSC-CM group.Cell immunofluorescence assay showed that the VEGFR2 expression in LV-FGF-2+-hGMSC-CM group was also hi gher than that in the ECM group,hGMSC-CM group and LV-vector+-hGMSC-CM group.(2)Tube formation assay showed that.HUVECs were cultured for 24 hours,the LV-FGF-2+-hGMSC-CM group had longer total tube length and total branching length than hGMSC-CM group and LV-vector+-hGMSC-CM group.(3)Transwell migration assay showed that the migrating cells number in the experimental group was significantly more than that in the empty vector group,normal hGMSCs group and negative control group.5.The results of improved CD31 immunohistochemistry showed that after 7 days of matrigel injected subcutaneously into the back of mice,the CD31 positive expression of matrigel plug in LV-FGF-2+-hGMSC-CM group was higher than that in hGMSC-CM group.ConclusionsOverexpression of FGF-2 gene promotes hGMSCs paracrine of angiogenesis-related growth factors,thereby obtaining an optimized conditioned medium for angiogenesis promotion.