Experimental Research On Culture And Identification Of Endothelial Progenitor Cells From Rat Peripheral Blood

Background and ObjectiveEndothelial progenitor cells are multipotent stem cells which could renew itself,proliferate and differentiate into mature endothelial cells.It is not only involved in the early embryonic blood vessel formation, but also closely related to neovascularization and angiogenesis in adult individuals. In addition to angiogenesis and repair of vascular injury, it also secretes a variety of growth factors to promote angiogenesis, such as epidermal growth factor (EGF), fibroblast growth factor 4,9 (FGF-4,9), vascular endothelial growth factor (VEGF), etc.In recent years, endothelial progenitor cells show a wide range of application in the repair of injured blood vessels,such as cardiovascular and cerebrovascular disease, diabetic retinopathy and wound healing,etc.Establishing the method of obtaining peripheral blood EPCs will lay a good foundation for the wide application of endothelial progenitor cells as seed cells for vascular tissue engineering.MethodsIn this study,mononuclear cells obtained from peripheral blood of rat were isolated by using density gradient centrifugation.They were divided into 4 groups by different culture conditions:group 1:without FN precoating, using the M199 basal medium(basal medium containing M199,10% fetal bovine serum);group 2:without FN precoating, using the M199 induction medium (induction medium containing M199,10% fetal bovine serum,VEGF10ng/mL,bFGF4ng/mL).group 3:FN precoating, using the M199 basal medium;group 4:FN precoating, using the M199 induction medium.Then they were cultured under different conditions in vitro to compare the differences of the growth.We recorded the number and the clonogenicity of the cells on the 7th day,calculated the numbers by SPSS-a kind of statistics software, and then checked wether the differences among the 4 groups had statistics significance or not. At last, we conducted immunohistochemistry and immunofluorescence identification. ResultsThe mononuclear cells from rat peripheral blood grew in the manner of keeping close to the wall in vitro,and grew slowly in the first 3-4 days.About 4 days later, the logarithmic growth phase appeared. 10days after the culture,the cells covered the bottom of the culture-bottle.The cells and cell colonies cultured for 7 days hinted:under the same culture conditions (we compared group 1 with group 3, group 2 with 4 group), precoating FN was benefit for the adherent proliferation of EPCs (t= 4.43,P<0.05; t=3.70, P<0.05).Excluding the impact of FN (we compared group 1 with 2, group 3 with group 4), we found that growth factors could promote mononuclear cells differentiation to EPCs, indicating that growth factors enhance proliferation of EPCs(t=-13.22, P<0.01;t=-10.96, P<0.01).At different times,CD34,Flk-1 and CD 133 showed positive expression at different rates.ConclusionsMononuclear cells isolated from rat peripheral blood by using density gradient centrifugation could be cultured and then differentiated into endothelial progenitor cells.FN is conducive to adhesion and proliferation of EPCs.VEGF and bFGF play an important role in the differentiation of EPCs.The success culture of EPCs in vitro will provide a sufficient number of seed cells for its application in vascular tissue engineering and offer new ideas for peripheral blood stem cell transplantation for the treatment of various diseases.