Effect Of Conditioned Medium Of Endothelial Progenitor Cells On Differentiation Of Mesenchymal Stem Cells Into Endothelial Cells

ObjectiveTo investigate the effects of mouse bone marrow-derived endothelial progenitor cells(EPCs)conditioned medium(CM)on the in vitro tube formation and differentiation into endothelial cells(ECs)of homologous mesenchymal stem cells(MSCs).Methods1.C57B/L6 mouse bone marrow cells of about 5 weeks old were used to obtain bone marrow cells,MSCs and EPCs were obtained by whole bone marrow adherence method and differential adherence method,respectively.2.Flow cytometry was used to detect the expression of surface markers of MSCs and EPCs,respectively.3.Functional identification of EPCs by dual fluorescent staining and in vitro tube formation experiments.4.The experiments were divided into groups: 0% EPCs-CM group(control group,whole LG-DMEM culture),25% EPCs-CM group(using 25% EPCs-CM 75% and LG-DMEM culture)and 50% EPCs-CM group(using 50% EPCs-CM and 50% LG-DMEM culture),real-time quantitative PCR(qRT-PCR)was used to detect the mRNA expression of CD31,vWF and eNOS in each group of MSCs at 1 week,2 weeks and 3 weeks respectively.5.According to the results of qRT-PCR,the experiments were subdivided.The 50% EPCs-CM group was used as the experimental group and the 0% EPCs-CM group was used as the control group.The expression of CD31 and CD34 in each group was detected by immunofluorescence.The in vitro tube-forming ability of each group of cells was examined.The NO content in each group of supernatants was detected by NO kit.Results1.Flow cytometry results showed that the positive expression rate of Sca-1 in the third generation MSCs was 93%,and the positive expression rates of CD34 and CD11 b were 18.2% and 31.9%,respectively.;The positive expression rates of EPCs CD34,CD133 and VEGFR2 were 91.8%,67.4% and 98.9%,respectively.2.EPCs can phagocytose Dil-labeled acetylated low-density lipoprotein(Dil-AC-LDL),which makes the cells red-fluorescent and binds to lectin(UEA-1)to make the cells green.A lumen-like structure can be formed on the Matrigel.3.qRT-PCR showed the expression of CD31,vWF and eNOS mRNA in 25% EPCs-CM concentration group at 3 weeks(1.45±0.81;1.47±0.92;3.79±0.85).The expression levels of vWF and eNOS mRNA were significantly higher than those in the control group.The difference was statistically significant(P<0.05);the expression of CD31,vWF and eNOS mRNA in the 50% EPCs-CM concentration group(2.28±0.25,1.76±0.71,8.27±1.65)were significantly higher than the control group.Statistically significant(P < 0.05).4.The results of immunofluorescence showed that the expression of CD31 and CD34 on the surface of MSCs in the experimental group was significantly higher than that in the control group;the cell formation ability of the experimental group was significantly stronger than that of the control group;The NO content in the cell culture supernatant of the control group was(8.81±3.41)?mol/L.The content of NO in the culture supernatant was(20.93±9.47)?mol/L,which was significantly higher than that of the control group,and the difference was significant(P<0.05).Conclusion1.EPCs conditioned medium can enhance the ability of MSCs to form tubes in vitro;2.EPCs conditioned medium can promote the differentiation of MSCs into endothelial cells(ECs)in vitro.