ABSTRACT
Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.
Subject(s)
Animals , Mice , Antigens, CD34 , Genetics , Metabolism , Antigens, Ly , Genetics , Metabolism , Cell Differentiation , Genetics , Physiology , Cell Line , Embryonic Stem Cells , Cell Biology , Metabolism , Epidermal Growth Factor , Pharmacology , Flow Cytometry , Gene Expression Regulation, Developmental , Hepatocyte Growth Factor , Pharmacology , Liver , Cell Biology , Metabolism , Membrane Proteins , Genetics , Metabolism , Mice, Inbred BALB C , Microfilament Proteins , Metabolism , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells , Cell Biology , Metabolism , Time FactorsABSTRACT
Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.
ABSTRACT
<p><b>OBJECTIVE</b>To observe calf thymus DNA damage induced by potassium dichromate in combination with glutathione (GSH).</p><p><b>METHODS</b>Atomic force microscope and ultraviolet spectrum (UV) were used to observe the alterations of the DNA ultrastructure and absorption spectrum.</p><p><b>RESULTS</b>Atomic force microscopy revealed no breaks of the DNA strand in response to treatment with potassium dichromate alone, but when coupled with GSH at proper concentrations, potassium dichromate induced alterations in the DNA structure and DNA fragmentation. UV examination also confirmed these findings by showing increased absorption intensity of the maximum UV peak following combined treatment of the DNA with potassium dichromate and GSH.</p><p><b>CONCLUSION</b>These morphological and spectrographic evidences verified the important role of GSH in mediating the generation of various tumor-inducing intermediate products of potassium dichromate.</p>
Subject(s)
Animals , Cattle , DNA , Chemistry , Genetics , DNA Damage , DNA Fragmentation , Glutathione , Toxicity , Microscopy, Atomic Force , Methods , Nucleic Acid Conformation , Potassium Dichromate , Toxicity , Spectrophotometry, UltravioletABSTRACT
Human CD34(+) hematopoietic cells, a distinctive cell population containing hematopoietic stem/progenitor cells (HSPC), have the capability to highly self-renewal, differentiation into all lineages of committed progenitor cells and reconstitution of both long-term hematopoiesis and immunefunctions after transplantation. CD34(+) hematopoietic cells from bone marrow (BM) recently have been employed for treating neoplastic and genetic disorders. This study was aimed to investigate membrane surface ultrastructures of bone marrow CD34(+) cell from mormal persons and leukemia patients and to compare their morphologic differences by using atomic force microscope (AFM). BM was collected from 5 normal donors and 6 leukaemia patients. All samples were layered on Ficoll-Paque gradients (specific gravity 1.077 g/ml) to separate the mononuclear cells. After that CD34(+) cells were purified by immuno-magnetic bead separation and evaluated with a FACS Calibur, these cells were detected by AFM of tapping mode inair. At lest 20 cells per samples were observed. The results showed that most of CD34(+) hematopoietic cells were like circle plate, the diameter was 10 - 14 microm. The surface of CD34(+) hematopoietic cell membrane was comparatively complex. The surface of CD34(+) hematopoietic cell membrane appeared as granular, with packed particles. With the region analysis function of IP2.1 software, the region of 2 microm x 2 microm was selected and four parameters of the surface (maximum peak-to-valley distance, average roughness, root-mean-squared roughness and mean height) were measured. Values of the 4 parameters showed that the characteristic parameters of CD34(+) HSPC from leukaemia were higher than that from normal person. It is concluded that AFM has specific advantages in analyzing cell membrane in the nanometer level and can gain more information. With the help of analysis software, AFM can be a helpful tool for fast leukaemic diagnosis and CD34(+) hematopoietic cells selection.