Preclinical Study Of The Treatment Of Hepatic Failure By Umbilical Cord Mesenchymal Stem Cells Transplantation

Part one Isolation and culturing of umbilical cord mesenchymal stemcells from transgenic EGFP C57BL/6J mice and identification of EGFPgeneObjectiveTo explore isolation, cultivation and proliferation of mice umbilical mesenchymalstem cells(UMSCs); to prove that the UMSCs isolated from transgenic EGFP C57BL/6Jmice can stably express enhanced green fluorescent protein (EGFP) in vitro, then theUMSCs labeled with EGFP can be observed in vivo.MethodsThe UMSCs were isolated from transgenic EGFP C57BL/6J mice on16th day ofpregnancy by collagenase and trypsin. The UMSCs were cultured and purified by adherentmethod. The morphological and growth condition of cells were observed. Cell surfacemarkers of the UMSCs were analyzed by flow cytometry. Differentiate potency ofUCMSC to osteogenic cells and adipocytes were evaluated. The expression of EGFP incells of different generations was examined by fluorescence microscopy, PCR andimmunocytochemistry.Results1. UMSCs were harvested successfully from mice umbilical cord by collagenase andtrypsin. The growth and proliferation of the cells in vitro were stable.2. The adherent cells displayed spindle shape, bundle-intensive parallel or swirlingarrangement were observed under inverted microscope.3. Flow cytometric analysis showed that the UMSCs expressed CD90and CD106, but did not express CD34and CD45.4. UMSCs had the ability to differentiate into the osteogenic cells and adipocytes.5. The expression of EGFP in the cells was observed by fluorescence microscopy.6. The results obtained using PCR showed that EGFP expressed in differentgenerations of the cells. Immunohistochemical staining showed that positive expressiveproducts appeared in cytoplasm.ConclusionsSuccessfully established the method of isolation, cultivation and proliferation of miceumbilical mesenchymal stem cells(UMSCs). The UMSCs isolated from transgenic EGFPC57BL/6J mice can stably express enhanced green fluorescent protein (EGFP) in vitro. Part two Umbilical cord mesenchymal stem cells contribute to thetreatment of Hepatic Failure in Mice and Cellular localizationObjectiveTo establish an experimental model of acute liver failure induced byD-galactosamine(D-GalN)/Lipopolysaccharide(LPS) in mice. To evaluate the therapeuticeffectiveness of UMSCs on acute liver failure. To observe the dynamic distribution ofUMSCs in vivo.MethodsMice were given an intraperitoneal injection of D-GalN/LPS，to induce acute liverfailure.6hours following model induction，0.4ml of UMSCs were injected through a tailvein in the experimental group and saline was injected in the same way in the controlgroup. The general situation, the recovery of liver function and the liver pathologicalchanges at different time points were compared between the two groups. Then collectedlivers and lung from the cells transplanted group at different time points, made frozensection and observed EGFP fluorescence labeled cells in the lung, liver tissue migrationand distribution patterns with fluorescence microscope. The amount of UMSCs of liver tissue from the cells transplanted group at different time points was analyzed by RelativeQuantification RT-PCR of the EGFP gene.ResultsThe survival rate was significantly higher in the cells transplanted group（40%） thanthat in the saline group（10%）. Liver function and the pathological injure of liver wereameliorated. Appreciable accumulation of transplanted cells with label were not found inthe liver at24h after transplantation, but occurred in the injured liver of the experimentgroup3days later. Relative quantitative RT-PCR showed that the expression of EGFP genein the liver of the experiment group3d,1month after transplantation were1.66times and5.17times compared with that of24h.ConclusionsThese findings demonstrated that the treating of mice acute liver failure by umbilicalcord mesenchymal stem cells is significantly effective. UMSCs labeled with EGFPtransplanted through tail vein can successfully homing and reside in injured liver．...