Study About The Therapeutic Effect Of Human Umbilical Cord Mesenchymal Stem Cell-secretion On Fulminant Hepatic Failure

Objective To investigate the therapeutic effect of human umbilical cord mesenchymal stem cell-paracrine substance on FHF rat, and to study the effect on hepatocytes proliferation and apoptosis.Methods Umbilical cord mesenchymal stem cells(UC-MSCs)were separated from human umbilical cord, and the surface makers of cells were detected by flow cytometry. Prepare human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM). FHF rat model was induced by intraperitoneal injection of D-galactosamine and they were randomly separated into three groups:. MSC-CM group, NS group, PHGF group.24 h later, lml MSC-CM, lml 0.9% NaCl solution and lml PHGF solution was injected into the penile vein of rats respectively. In each group (n=8 per group), blood samples were collected at 12,24,36 and 60h after treatment from inner canthus for analysis of blood ALT and TBIL levels. We used five rats per group for tissue collection after sacrifice at 36h after treatment for HE staining, PCNA immunohistochemical staining and TUNEL staining to detect the inflammatory cell infiltrate, hepatocyte proliferation and apoptosis. At the axenic condition, we collected the rat liver tissue and freezed in -80℃refrigerator, then extacted the RNA to detect the express of HGF, EGF, HB-EGF, TNF-a and OSM in rat liver by RT-PCR. At the same time, we collected the peripheral blood of rats to detect the content of TNF-a and IL-10 by ELISA. We closely observed and recorded the survival status and survival time of the surplus rats(n=10 per group) for the survival analysis.Results 1. Msenchycal stem cells were isolated from umibilical cord by collagenase and trypsin digestion sufficiently, then the cells were collected for the primary culture. 24 hours later, we can be observed adherent cells at the light microscope, the cell morphous was as fusiform shape, irregularity, different size, fibroblast-like, whirlpool-like growth. Flow cytometry showed that the cells with high expression of CD29, CD49, CD90, CD 105, and HLA-I, with low expression of hematopoietic cell phenotype CD34, CD45 and endothelial cell phenotype CD31, low expression of CD14, HLA-DR.2.24h after treatment, the levels of ALT and TBIL in MSC-CM and PHGF groups were lower than NS group (P<0.05), but there was no significant difference between MSC-CM and PHGF group.3. The ELISA assay showed that the level of TNF-a in MSC-CM and PHGF groups were lower than the NS group (P< 0.05), however, the level of IL-10 were higher than the NS group (P<0.05), there was no significant difference between MSC-CM and PHGF group.4. The PCNA staining showed that the PCNA-positive hepatocytes in MSC-CM and PHGF groups were more than NS group (P< 0.01), but no significant difference between MSC-CM and PHGF group; the TUNEL staining showed that the TUNEL-positive hepatocytes in MSC-CM and PHGF groups were less than NS group (P<0.05), but there was no significant difference between MSC-CM and PHGF group.5. We found that the expression of the 5 genes in MSC-CM and PHGF groups were approximately increased 2-4 folds versus NS group (P<0.05), there was no significant difference between MSC-CM and PHGF group.6. Survival analysis found that the survival rate of MSC-CM and PHGF groups were higher than NS group (P<0.05), but no significant difference between MSC-CM and PHGF group.Conclusions We could be harvested a large number of cells by collagenase digesting method. The cells have strong proliferation ability and rapid expansion, its morphology and immunophenotype are likely to BM-MSCs. It is consistented with the biological characteristics of MSCs, provided a reliable cells source to further study. The paracrine substance of human umbilical cord mesenchymal stem cells can stimulate hepatocytes proliferation, inhibit hepatocytes apoptosis, improve liver function and survival of FHF rats, potentially creating a new avenue for the treatment of FHF.