Combination Of Fucosylation And CD26Inhibition Enhances Human Cord Blood Stem Cells Engraftment And The Study Of Interactions

Object: The study was aimed to explore the usage of glycosylation repaired by FTVIcombined with CD26inhibition repaired by Diprotin A as a method of improvingengraftment efficiency of CB CD34+hematopoietic stem cells(expressing CD45)and.Meanwhile we purposed to explore the effects the effect and interactions ofglycosylation engineering and inhibition of CD26in vivo.Method: Obtained purified CD34+stem cells from cord blood by immunomagneticbeads method.We used flow cytometry to detect the expression of E、P-selectin ligandrepaired by glycosylation engineering,used flow cytometry to detect the CD26repairedby CD26inhibition and microplate spectrofluorometer to detect the activity of CD26repaired by CD26inhibition.Purified CB-derived CD34+cells received sham treatment，fucosylation,CD26inhibition,fucosylation/CD26inhibitor (combined).Cells were thenintravenously infused into sub-lethally irradiated NOD/SCID mice,respectively.Toinvestigate the effect of engraftment in vivo, we observed survival rate and monitoredthe expression of human CD45in peripheral blood after transplantaton to six weeks byflow cytometry.Meanwhile we used microplate spectrofluorometer and flow cytometryto detected the interactions of glycosylation engineering and CD26inhibitionResult:1.The effects of glycosylation engineering and inhibition of CD26ex vivo:Theexpression of P-selectin ligand on the surface of cord blood stem cell was promted from(75.19±1.15)%to (94.11±1.21)%after glycosylation engineering.The expression ofE-selectin ligand on the surface of cord blood stem cell was promted from （25.01±1.00）%to（53.52±1.17）%after glycosylation engineering.The expression ofCD26on the surface of cord blood stem cell was no obvious change from（10.92±1.52）%to （9.88±0.95）%but the activeness of CD26was loss after CD26inhibitor.2.The effects of transplantation:Starting from two weeks after transplantation,CD45was detected0.45±0.78%in the peripheral blood of recipients receivingcombined treatment, but not in other groups. Fourth weeks after transplantation wedetected（0.73±0.22）%（,2.95±0.12）%（,3.21±0.43）%and（8.87±0.83）%in peripheralblood of sham control， fucosylation, CD26inhibition, and combined groups,respectively. Fourth week after transplantation, we observed （12.87±0.62）%,（29.41±0.21）%（,32.76±0.42）%and（41.07±0.32）%of CD45expression in peripheralblood of control, fucosylation,CD26inhibition,and combined groups, respectively.Allsub-lethally irradiated NOD/SCID mice received physiological saline injection not HSCtransplantation were dead in one week.The median survival of mice in experimentalgroups were more than6weeks.The peak mortality time was one week afterirradiation.The overall viability rate six weeks after transplantation for CD26inhibitionand combined groups was significantly higher than that for other three groups (40%forsham control，40%for fucosylation,80%for CD26inhibition, and80%for combinedgroup, P <0.05).3.The interactions of glycosylation engineering and inhibition of CD26ex vivo:The expression of P-selectin ligand on the surface of cord blood stem cell wasno obvious change from (75.19±1.15)%to （74.01±1.27）%after CD26inhibitor.Theexpression of E-selectin ligand on the surface of cord blood stem cell was no obviouschange from （25.01±1.00）%to （25.50±1.41）%after CD26inhibitor.The expressionof CD26on the surface of cord blood stem cell was no obvious change from（10.92±1.52）%to（10.44±1.16）%after glycosylation engineering.The the activenessof CD26was no obvious change from65.34U to65.68U after glycosylationengineering. Conclution:These results provide evidence that combined treatment significantlyenhances the speed of early engraftment of CB-derived HSPC and improve the overallsurvival rate of transplanted receipient mice.Our results may lead to a new method toimprove the utility of CB-derived HSPC in hematopoietic stem cell transplantation.Glycosylation engineering and CD26inhibitorcan playing role in different targets inhoming are effective in vitro experiment but there is no evidence which proved thesynergy effectiveness of combination.