ABSTRACT
AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. METHODS: EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin beta1), CD49f (integrin alpha6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class I (A, B, C) and class II (DR) expression was studied by flow cytometry only. RESULTS: FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class II (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. CONCLUSION: Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.
Subject(s)
Fetal Stem Cells/metabolism , Hepatocytes/metabolism , Liver/metabolism , Albumins/metabolism , Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Cells, Cultured , Epithelial Cell Adhesion Molecule , Female , Fetal Stem Cells/immunology , Flow Cytometry , HLA Antigens/metabolism , Hepatocytes/immunology , Humans , Immunohistochemistry , Immunomagnetic Separation , Integrin alpha6/metabolism , Integrin beta1/metabolism , Keratins/metabolism , Liver/embryology , Liver/immunology , Phenotype , Pregnancy , Pregnancy Trimester, Second , Reverse Transcriptase Polymerase Chain Reaction , Thy-1 Antigens/metabolism , alpha-Fetoproteins/metabolismABSTRACT
The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m2 which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2, whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m2. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2.
Subject(s)
Hepatocytes/metabolism , Hepatocytes/radiation effects , Animals , Cell Survival/radiation effects , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Radiation , Goats , Hepatocytes/cytology , Hepatocytes/transplantation , In Vitro Techniques , Inactivation, Metabolic/radiation effects , L-Lactate Dehydrogenase/metabolism , Lipid Peroxidation/radiation effects , Transplantation, Heterologous , Ultraviolet RaysABSTRACT
Ultraviolet-B (UV-B) irradiation in the range of 280-320nm has shown to be a promising immunomodulatory tool in xenogenic hepatocyte transplantation. Most of the studies documenting the effect(s) of UV-B irradiation on hepatic transplantation have been carried out in small model systems with very little information available in larger animals. The aim of the present investigation was to study in vitro the effect(s) of UV-B irradiation (302 nm) at 0, 250, 500, 1250 and 2500 J/m2 on the viability and cellular responses in the isolated goat hepatocytes. The results showed that the cells irradiated at 0, 250, 500, 1250 and 2500 J/m2 demonstrated a viability of 90-95%. However, intracellular [Ca2+]i influx as quantitated by Flu 3-acetete showed a significant increase with irradiation as observed in confocal microscope. The intracellular pH (quantitated by the flourescence of BCCEF) although tend to show an increase with UV-B irradiation was not statistically significant. The present observations suggest that there is a modulation in the intracellular [Ca2+]i concentration within the hepatocytes at higher dose of UV-B irradiation without altering the viability of hepatocytes. These observations are significant for the xenotransplantation of cells.
