The Study Of Homing Mechanism For Endothelial Progenitor Cell Recruitment To Mouse Liver With Fibrosis And Cell Tracking By In Vivo 3.0-T MR

Partâ… Isolation,culture and identification of bone marrow-derived endothelial progenitor cells of C57 mouseObjective:To investigate the methods of isolation,culture and identification of endothelial progenitor cells(EPCs) from C57 mouse bone marrow,as well as observe the cell marker expression during proliferation and differentistion in vitro.Materials and methods:The mononuclear cells were isolated from C57 mouse bone marrow(BM) by density gradient centrifugation and were cultured in vitro via adhesion selection methods.Then the BM derived mononuclear cells were cultured in M199 medium supplemented with vascular endothelial growth factor(VEGF, 20ng/ml),basic fibroblast growth factor(bFGF,1ng/ml) and 20%fetal bovine serum (FBS).Attached spindl-shaped cells were detached at day 7,and then identified by endothelial specific markers.The expression of Sca-1 and flk-1(VEGFR-2) were detected by flow cytometry.Endothelial function was determined by cell absorption of DiI complexed acetylated low-density lipoprotein(DiI-acLDL) and fluorescein labeled ulex europaeus agglutinin-1(FITC-UEA-1),and then scanned by fluorecent inverted microscope system to confirm EPCs lineage.In vitro vasculogenesis activity was assayed by in vitro vasculogenesis kit.Results:The cultured BM-derived mononuclear cells began to adhere to the wall at day 1.The adherent cells stretched and exhibited the clone-like morphology after 4d cultivation.The cells proliferated faster after 7d.Flow cytometry analysis demonstrated that the Sca-1⁺/Flk-1⁺double positive cells account for 67.59±5.3%.The cells could take up DiI-acLDL,and bind to FITC-UEA-1,showed double positive fluorescence under fluorescent iverted microscope and the positive rate was 89.03±6.11%.The cultured BM-derived MNCs showed can form micro-vascular structures on the surface of ECMatrix^TM.Conclusion:Relatively purified EPCs derived from C57 bone marrow could be obtained by the procedrue of isolation,culture and exhibited some of the characteristics and functions of endothelial cells(ECs) after 7d inducing culture in vitro.Partâ…¡The significance of stromal-cell derived factor 1α(SDF-1α)/CXC chemokin receptor 4(CXCR4) in endothelial progenitor cells recruiting to liver with fibrosisObjective To detect the genes expression of SDF-1α/CXCR4 of liver with fibrosis induced by CCl4,to evaluate the association between SDF-1α/CXCR4 axis and EPCs homing to liver with fibrosis and benefit for further cell therapy.Materials and methods:Liver fibrosis was induced in C57 mice(female,six weeks old) by intraperitoneal injection with 40%CCL4/olive solution(1ml/kg,twice one week,4 weeks).The gene expression of SDF-1αand CXCR4 were detected in livers with fibrosis and normal livers.The SDF-1αwas detected with RT-PCR and immunohistochemistry and CXCR4 was detected with RT-PCR and Western-blotting. The migratory capacity of BM-EPCs of each group was evaluated by Transwell chamber and the expression of CXCR4 on EPCs was detected by FACS.Results:The expression of SDF-1αand CXCR4 was significantly increased in liver with fibrosis compared to normal liver(P＜0.05).The positive rate of CXCR4 expression on BM-EPCs by FACS was 69.35%±16.04%.The number of BM-EPCs increased obviously with SDF-1α(from 87.80±14.24/high power field to 158.40±12.66/high power field,P＜0.01).After administration of AMD3100,which specifically blocks binding of SDF-1αto its endogenous receptor CXCR4,the number of BM-EPCs migrating to SDF-1αwas reduced from 158.40±12.66/high power field to.92.60±13.59/high power field(P＜0.01).Conclusion:SDF-1α/CXCR4 axle plays an important role in EPCs homing to liver with fibrosis,which provides a rationale for treatmentPartâ…¢R2^* and R2 mapping by clinical 3.0-T MR for quantifying recruitment of SPIO labeled endothelial progenitor cells to liver with firbosis in vivoObjective:To prospectively evaluate R2^* and R2 mapping by clinical 3.0-T MR for tracking and quantifying recruitment of iron oxide(SPIO)-labeled endothelial progenitor cells(EPCs) to injury liver in vivo.Materials and methods:All animal experiments were approved by the institutional animal care committee.The bone marrow derived EPCs from C57 mice were isolated and cultured for 4 days and examined in vitro for lineage markers.Then the 4-day-culture cells were labeled with ferumoxides-protamine sulfate complex (FE-PRO).Iron uptake was analyzed with electron microscope and Prussian blue staining.Agarose gel phantoms containing different amounts of EPCs(0-2.0×10⁶ cells per milliliter of 1.0%agarose gel) were analyzed with clinical 3.0-T MR R2 and R2^* relaxometry.For in vivo tracking,liver fibrosis was induced in healthy C57 mice (female,six weeks old,19g-20g weight) by administration of 40%CCl4/olive by single intraperitoneal injection.Follow up serial R2^* and R2 mapping of livers with fibrosis and normal livers of C57 mice were measured with 3.0T MR at 2h,4d,8 d and 12d after EPCs transplantation.After 12 days,all mice were sacrificed,and the livers were histopathologically examed.Results:Electron microscope and Peals' Prussian blue stain revealed the efficiency of SPIO particle uptake was more than 95%and non structural changes of labeled cells compared with nonlabeled control cells.R2 and R2^* values were linearly correlated with number of iron-loaded cells in agarose gel phantoms(r value were 0.98 and 0.99,P＜0.01),R2^* effects were significantly greater than R2 effects for tagged cell phantoms(P＜0.01);MR cells tracking demonstrated that R2^* effects of livers in all groups were obviously greater than R2 effects(P＜0.01).The R2^* effect of livers with or without frbrosis were greater than control group at 2h,4d and 8d after intravenous administration of EPCs(P＜0.05).R2^* value gradually decreased with time.The R2^* effect of livers with frbrosis were greater than normal group at 2h,4d and 8d after intravenous administration of EPCs(P＜0.05).The R2 effect of livers with or without frbrosis were greater than control group at 2h,4d and 8d after intravenous administration of EPCs(P＜0.05).R2 value gradually decreased with time.The R2 effect of livers with frbrosis had no difference with normal group at different times after intravenous administration of EPCs(P＜0.05).R2^* values were higher than R2 values in all groups(P＜0.05).Conclusion:Both quantitative R2^* and R2 mapping provide useful methods for quantifying intravenous transplanted progenitor cells homing to liver and R2^* mapping is better than R2 mapping,which is an accurate and non invasive approach for clinical cell therapy.