The Improvement Of Placental Perfusion In Pre-eclampsia By Transplantation Of Endothelial Progenitor Cells

PartⅠThe Culture and Identification of Endothelial ProgenitorCells from Peripheral Blood of Pre-eclampsia PatientsObjective The purpose of this study was to investigate the methods of isolating,culturing and identifying endothelial progenitor cells from peripheralblood of pre-eclampsia patients and to set the scope for further researchinto the relationship between pre-eclampsia and endothelial progenitorcells.Methods Mononuclear cells were isolated from pre-eclampsia patient peripheralblood by ficoll density gradient centrifugation and re-suspended inM-199 medium supplemented with 20% fetal bovine serum and humanvascular endothelial growth factor,culturel dishes were coated with rattail gel before culture.7 days later,the cells were identified by positiveexpression of both CD34 and CD133 under fluorescence microscope andflow-cytometry and positive expression of factorⅧas shown byimmunocytochemistry.Results The results show that the number of mononuclear cells expressing doublepositive for CD34 and CD133 was(4.29±1.21) % when just isolated from peripheral blood,increasing to(79.13±4.08) % after beingcultured for 7 days,moreover,the cells showed that double positiveexpression of CD34 and CD133 was determined under fluorescencemicroscope and positive expression of factorⅧwas shown byimmuncytocheminstry.Conclusions It is concluded that endothelial progenitor cells can be isolated fromperipheral blood by ficoll density gradient centrifugation and cultured inM-199 medium supplemented with 20% fetal bovine serum and humanvascular endothelial growth factor,EPCs can be identified by positiveexpression of both CD34 and CD133 under fluorescence microscope andflow cytometry and positive expression of factorⅧas shown byimmunocytochemistry. PartⅡChanges in Number and Biological Function of EndothelialProgenitor Cells in Pre-eclampsia PatientsObjective The purpose of this study was to examine the changes in number andfunction of endothelial progenitor cells(EPCs) from peripheral blood(PB) in pre-eclampsia patientsMethods 20 women with pre-eclampsia and 20 normal pregnant women in the thirdtrimester were studied.Mononuclear cells(MNCs) from PB wereisolated by Ficoll density gradient centrifugation.EPCs were culturedaccording to the method introduced in partⅠand identified by positiveexpression of both CD34 and CD133 under fluorescence microscope,and positive expression of factorⅧas shown by immunocytochemistry.The number of EPCs was flow-cytometrically determined.Proliferationand migration of EPCs were measured by MTT assay and modifiedBoyden chamber assay,respectively.The adhesion activity of EPCs wasdetected by counting the number of the adherent cells.Results The results showed that,compared with normal pregnant women,thenumber of EPCs was significantly reduced when just isolated from PBand after being cultured for 7 days in pre-eclampsia patients.Thenumber was(4.29±1.21) % vs.(15.32±2.00) % and(79.13±4.08) % vs.(90.57±2.53) %,respectively.(P＜0.01),the functional activity of EPCsin pre-eclampsia patients,such as proliferation((13.45±1.68) % vs.(18.75±1.67) %),migration((37.25±7.28) cells/field vs.(67.10±9.55)cells/field) and adhesion activity((20.65±5.19) cells/fieldvs.(34.40±6.72) cells/filed) was impaired(P＜0.01).Conclusions It is concluded that the number and function of EPCs are significantlydecreased in pre-eclampsia patients. PartⅢAn Improved Method to Duplicate Rat Models forPre-eclampsiaObjective The purpose of this study was to duplicate a rat model for pre-eclampsiaby an improved method.Methods Female adult Wistar rats were randomly divided into 6 groups:normalpregnant group(group A),intraperitoneally infected with L-NAMEgroup(group B),intraperitoneally infected with saline solution group(group C),bilateral uterine artery ligation group(group D),shamoperation group(group E) and coarctation of abdominal aorta combinedwith intraperitoneally infected with L-NAME(group F),then,changesin systolic pressure,urine,pathologic injury in kidneys and placenta andthe weight of placenta,the fetal body and head were monitored.Results The treatment in group F resulted in a significant increase in maternalsystolic pressure and proteinuria in just gestational 13 days,thesymptoms were earlier than group B and E(P＜0.05),it also caused asubstantial decrease in the weight of the pups and the fetal head,accompanied by typical renal and placental pathological changes,thesesigns are similar to those of pre-eclampsia.Conclusions It is concluded that coarctation of abdominal aorta combined with intra-peritoneum infected with L-NAME is an ideal way to build the animalmodel of pre-eclampsia. PartⅣThe Improvement of Placental Perfusion in Pre-eclampsiaModel Rats by Transplantation of Endothelial Progenitor CellsObjective The purpose was to investigate whether the placental perfusion inpre-eclampsia model rats can be improved by transplantation ofendothelial progenitor cellsMethods The rat model of pre-eclampsia was induced by coarctation of abdominalaorta combine with intraperitoneal infected with L-NAME;endothelialprogenitor cells of rats were harvested from peripheral blood,after beingsigned with DiI,the cells were transplanted to the placenta of rats withpre-eclampsia.The micro vascular density was detected by positiveexpression of factorⅧas shown by immunohistochemistry,thegestational result was observed by measurement of the weight of ratfetus and placenta.The expression of nestin in rat fetal cerebrum wasdetected by western blotting.Results The results showed that,the cells were alive after being transplanted to thepalcenta.Compared with the control group,the MVD of thetransplantation group was increased.The expression of nestin wasweaker than untreated group.Conclusions It is concluded that placental perfusion in rats with pre-eclampsia canbe improved by transplantation of endothelial progenitor cells...