Intrahepatic Tracking Of Bone Marrow-derived Endothelial Progenitor Cell After Transplanting Into Rats With Liver Ifbrosis

OBJECTIVE Protracting pathogenetic condition of hepatic fibrosis, will cause functionalhepatocytes’ decreasing relatively, and meanwhile lead to overly depositing of extracellular matrix (ECM),it will cause narrow of sinusoidal shape gap and occlusion of small hepatic vascular, promoting hepaticcirrhosis. Currently studies have indicated that endothelial progenitor cells (EPCs) can participate in vesselsof newborn and repair. However, EPCs are almost isolated by healthy bone marrow or peripheral bloodnow, very few to study whether EPCs could get from diseases individual, thereby the clinical application ofautologous cell transplantation is limited. Here, we take liver-fibrosis rats as study object, isolating,inducing and training to their bone marrow-derived endothelial progenitor cells (EPCs), trying to providesuitable donor cells for the treatment of liver fibrosis and angiogenesis by auto-cellular transplant,meanwhile, established a method of PKH26-labeled bone marrow-derived endothelial progenitor cells(EPCs) into rats with liver fibrosis and observe cell immigration and differentiation in liver aftertransplantation. The study will provide theoretical basis and practical foundation for the clinical applicationin treating liver fibrosis.METHODS Built poison inducing liver-fibrosis rat mould by hypodermic injection carbontetrachloride(CCl4）, and separated mononuclearcell from bone marrow of liver-fibrosis rat by densitygradient separation then these cells were cultured and induced into EPCs in vitro by using the method ofdifferential adhesion. The phenotype and purity of EPCs were detected by flow cytometry. Thephagocytosis function of EPCs was identified by stainning with DiL-ac-LDL and FITC-UEA-1. Lumen ofblood vessel cavitation test identified its blood vessel function. Bone marrow-derived EPCs were isolatedand cultured from rats with liver fibrosis, and then labeled with PKH26in vitro. Under the scanningconfocal microscopy and flow cytometry, PKH26fluorescent labeling rate and cell survival rate weremeasured．The PKH26fluorescent labeling of EPCs were transplanted into rats with liver fibrosis via thetail vein, and then observed migration situation in the liver. Endothelial cell markers CD31and von willebrand factor (vWF) were detected by immunofluorescence.RESULTS By density gradient separation and differential adhesion method, it could successfullyinduce and differentiate EPCs with specificity phaenotype and function from liver-fibrosis rat. The“cobblestone” structure of EPCs was observed after14days’ culture. About63.9percent cells wereCDl33+/VEGFR2+EPCs. These cells have phagocytosis function possessed by mature endothelial cells,and could form primitive vascular tube-like structures when planted in matrigel. The PKH26-labeled EPCsappeared red fluorescence and the labeling rate was96.65percent. Compared with unlabeled cells, thelabeled cells in good condition, no significant changes in the growth curve. After transplanted into hepaticof rats, the PKH26-labeled cells were mainly distributed in blood vessel endothelium along fibers andhepatic sinusoids in hepatic lobule and expressed endothelial cell-specific antigen such as CD31and vWF.Cue exogenous EPCs had proneness of moving to endogenous vessel wall, it established the base of bloodvessel’s repair and reconstitution.CONCLUSION By the methods of density gradient separation with differential adhesion, bonemarrow cells in rats with liver fibrosis could be successfully induced and differentiated into EPCs withcharacteristic phenotypes and functions. PKH26could be used to label and track EPCs in the liver of ratswith liver fibrosis in vitro. The PKH26-labeled cells may migrate to the liver vascular and differentiate intomature endothelial cells.