Bispecific Antibody Promote Rat Bone Marrow-derived Endothelial Progenitor Cells Homing And Angiogenesisi Efficiency On Ischemia Myocaidium

PART I Isolation, Culture and Identification the ratEndothelial Progenitor CellsObjective: To establish a stable approach of isolation, culture andidentification the rat endothelial progenitor cells, also explore theirdifferentiation conditions.Methods: Bone marrow mononuclear cells were extracted from ratfemoral and tibial bone marrow by the means of percoll density gradientcentrifugation, plated on culture flask coated with fibronectin, then inducedculture with bFGF, EGF, VEGF for14days. Cellular immunocytochemistrywas performed to identify the cell surface markers, laser scanning confocalmicroscopy was performed to detect the adherent cells which stained withFITC-UEA-1and Dil-acLDL. Results: The adherent cells could show the typical whirllikearrangement and paving stones growth. EPCs showed CD31、CD133、CD34、vWF、Flk-1positive at different times, also evidenced FITC-UEA-1and Dil-acLDL double fluorescence staining positive.Conclusions: Bone marrow mononuclear cells could be extractedfrom rat by use of a density gradient centrifugation on percoll, then we couldgain high purity EPCs after induction culture with bFGF, EGF, VEGF,which has identical features with the endothelial cells. PART II Bispecific antibody efficiency of rat bonemarrow-derived endothelial progenitor cells transplantationfor myocardial angiogenesisObjective: To investigate the effect of bone marrow-derived endothelialprogenitor cells (EPCs) transplantation for myocardial angiogenesis with the support of bispecific antibody (BiAb).Methods: Isolation, culture and identification of EPCs from rat bonemarrow. BiAb （CD34×AMLCA） was prepared by chemicalheteroconjugation and armed with EPCs. Rat with infracts creating byligation of the left anterior descending artery (LAD) were injected via the tailvein．This study included four groups: EPCs+BiAb, EPCs, BiAb, andcontrol groups. Five weeks after treatment, calculated left ventricularejection fraction (LVEF) and left ventricular fractional shortening (FS) toevaluate cardiac function. The expression level of VEGF was discoverd bythe means of fluorescent quantitative PCR and western blot.Results: Compared with other groups, EPCs+BiAb group showdimproved cardiac function(P<0.05), increased VEGF levels (P<0.05) andhighest homing number of EPCs (P<0.05).Conclusion: Combination of bispecific antibody could increase thehoming rate of EPCs in ischemic myocardium, improve the cardiac functionand promote the myocardial angiogenesis.