Effects Of Different Doses Of Puerarin On Proliferation And Migration Of Vascular Endothelial Cell From Human Umbilical Cord Blood

Objectives:To study the effect of different doses of puerarin on proliferation and migration of vascular endothelial cell(EPCs) from human umbilical cord blood and compare with the physiological dose of estrogen(E2).Methods:(1) Separates the mononuclear cells, human umbilical cord blood were collected from placenta of healthy women with term pregnancy, The mononuclear cells(MNCs) were isolated by gelatin precipitation and density gradient centrifugation; (2)Induction of differentiation:The cells were plated on culture flask(4μg/cm2),collect the non-adherent cells after 24 hours to cultivate to the fourth days, washed out non-adherent cells with phosphate buffer, changed the culture medium and continue to cultivate to the seventh day, further washed out the non-adherent cells with phosphate buffer, remain adherent cells for experimental use.(3)EPCs identification:the attached cells after 7 days were stained with DIL-LDL,FITC-UEA-1,CD 133,and observed by fluromicroscope. (4)Proliferation and migration test:1)Cell proliferation test (WST),manual counting and flow cytometry were performed with 7days EPCs in response to different doses of puerarin and E2 to investigate the proliferative effects.2). EPCs were seeded to the the Coster Transwell,600ul culture medium with different doses of puerarin and E2 were added into lower layer. Manual counting the number of EPCs migrated toward lower layer using high power microscope.Results:(1)Attached cells were observed after 4 days and cluster growth were noticed after 7days, it can be seen more blood island-like cell clusters after 9 days. More than 90% attached cells were double positive immunofluorescence. (2)WST showed that puerarin can promote the proliferation of EPCs presenting concentration-dependent, which shows significantly statistically significance (P<0.05) compared each group and with control group; E2 also shows significantly statistically significance compared with the control group and each dose of puerarin. (3)Manual counting test showed the cell numbers were increased by different doses of puerarin, the cell number, which shows significantly statistically significance (P<0.05) compared each group and with control group; E2 also shows significantly statistically significance compared with the control group and each dose of puerarin. (4).Cell cycle analysis:EPCs was increased S phase in response to puerarin, which shows significantly statistically significance (P<0.05) compared each group and with control group; E2 also shows significantly statistically significance compared with the control group and each dose of puerarin. (5)Transwell assay revealed that puerarin enhanced EPCs migration, which shows significantly statistically significance (P<0.05) compared each group and with control group; E2 also shows significantly statistically significance compared with the control group and each dose of puerarin.Conclusion:The proliferation and migration can be enhanced by Puerarin, and the numbers of EPCs in S phase can be increased,which effects shows concentration-dependent and is weaker than E2...