MiR-210 Promotes The Expression And Secretion Of Vascular Endothelial Growth Factor In BMMSCs

Objective To investigate the effect of mi R-210 on the expression and secretion of vascular endothelial growth factor(VEGF)in bone marrow mesenchymal stem cells(BMMSCs),which may provide experimental evidence for the application of micro RNA on the bone vascularizationin clinics.Methods The mouse BMMSCs were cultured in vitro by whole bone marrow adherence method,and to construct the lentiviral vector using Lenti-Lac Z and Lenti-mi R-210 respectively.The mouse BMMSCs and human umbilical vein endothelial cells(HUVEC)were transfected respectively.MTT assay to detect the proliferation of mouse BMMSCs.The expression of VEGF m RNA in Lenti-mi R-210 / BMMSCs and Lenti-Lac Z / BMMSCs was detected by RT-PCR after lentiviral vector transfection on the 7th day.The expression of VEGF m RNA was detected by Western blot after 14 days.The expression of Lenti-mi R-210 / HUVEC and Lenti-Lac Z / HUVEC was detected by PKH26 red fluorescent dye.The effect of the two groups was observed by observing the effect of tube formation on the 24 h Matrigel test by fluorescence microscopy;Constructing Hy Stem-HP gel sustained-release system,BMMSCs composited with agomir-210 and agomir-NC were combined with Hy Stem-HP gel to inject subcutaneously in nude mice.After 7 days,all the specimens were fixed in 10% neutral formalin and embedded in paraffin after operation,then stained with CD31 immunofluorescence and CD31 positive cells were detected.Statistical analysis was performed using SPSS 13.0 software.The experimental data were presented as mean ±standard deviation.The experimental data used t test and variance analysis.The testlevel was ? = 0.05.Results After 5-7 days of whole bone marrow culture,some cells were spindle or polygonal,and scattered in the distribution.The adherent cells were spindle-shaped spiral growth after passage,and cell morphology gradually consistent with the passage of culture.Mi R-210 lentiviral vector was transfected into mouse BMMSCs and HUVEC.When MOI = 10,the transfection efficiency was the highest.MTT assay showed no effect on the proliferation of BMMSCs.LMR-210 and Lenti-Lac Z cells were cultured after 7 days,and the expression of VEGF m RNA was detected by RT-PCR.,Mi R-210 promoted the expression of VEGF in mouse BMMSCs(P <0.01).Western blot was used to detect the expression of VEGF protein in the two groups in the 14 th days,the expression of VEGF protein in mi R-210 group was significantly higher than that in control group,which was consistent with RT-PCR test.Lenti-mi R-210 group compared with Lenti-Lac Z in 24 h Matrigel tube formation test results in more obvious annular tubular structure,more end-end contact,and more complete tube wall,the length of tubules was statistically significant(P <0.05).Agomir-210 and agomir-NC with BMMSCs Hystem-HP gel sustained-release system injected subcutaneously in nude mice for 7 days,CD31 immunofluorescence staining showed that the content of CD31 positive cells in agomir-210 group was significantly higher than that in agomir-NC group,which was about 3 times that of agomir-NC group.Conclusion The expression and secretion of VEGF in mouse BMMSCs can be effectively promoted by transfection of target gene mi R-210 through lentiviral vector.The subcutaneous experiment of agomir-210 Hy Stem-HP gel in nude mice showed that agomir-210 promote BMMSCs induced-angiogenesis.