ABSTRACT
OBJECTIVE@#To investigate the Methods for culturing two types of endothelial progenitor cells (EPC) from human umbilical cord blood and study their differentiation traits and the depressant effect of asymmetric dimethylarginine (ADMA) on its proliferation.@*METHODS@#Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch(HES) and density gradient centrifugation.Isolated cells were cultured in the medium supplemented with vascular endothelial growth factors (VEGF) and basic fibroblast growth factors (bFGF). The growth characteristics and biological features of the cells were observed at different time points and identified by morphology,immunofluorescence staining,reverse transcription polymerase chain reaction (RT-PCR), and flow cytometry.Attached cells were incubated with different concentrations of ADMA (1,5, and 10 micromol/L) for 24,48, and 72 hours. Methylthiazoletetrazolium (MTT) assay and quantified colony forming units (CFUs) were used to assess the proliferation of endothelial progenitor cells.@*RESULTS@#The attached cells were divided into 2 types:early EPC and late EPC. Early EPC changed from small sized round cells to spindle shaped cells and late EPC formed a typical cobblestone-like cells. Fluorescence microscopy showed that EPC were positive for both Dil-acLDL uptake and FITC-UEA-I binding.RT-PCR and FACS showed the difference of endothelial cell-specific,gene expression and changed AC133,CD34, and KDR among different times.Incubation of EPC with ADMA dose and time-dependently decreased the number and the proliferation of EPC.@*CONCLUSION@#There are 2 types of EPC from a source of human umbilical cord blood and ADMA may depress the EPC proliferation, providing a basis for further research.
Subject(s)
Humans , Arginine , Pharmacology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Culture Media , Depression, Chemical , Endothelial Cells , Cell Biology , Fetal Blood , Cell Biology , Leukocytes, Mononuclear , Cell Biology , Stem Cells , Cell BiologyABSTRACT
OBJECTIVE@#To investigate the effect of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, on the activation of hepatic stellate cells (HSCs) and its mechanism.@*METHODS@#Primary HSCs isolated from SD rats were cultured and treated with different concentrations (1, 3 or 10micromol/L) of ADMA for various periods (12 approximately 48h). Expression of alpha-smooth muscle actin (alpha-SMA) and synthesis of type-I collagens in HSC were determined. Messenger RNA levels of the transforming growth factor-beta1 (TGF-beta(1)) in the HSCs were determined using RT-PCR. Intracellular reactive oxidant species (ROS) production was measured using oxidant-sensitive fluorescent indicator. Activation of nuclear factor-kappaB (NF-kappaB) was detected by electrophoretic mobility shift assay (EMSA).@*RESULTS@#ADMA could increase alpha-SMA-positive cells ratio and Type I collagens production of HSCs in a concentration- and time-dependent manner, concomitant with the increase of the TGF-beta(1) mRNA level. Treatment with ADMA (10micromol/L) significantly increased the intracellular ROS production and activated NF-kappaB. Such effects of ADMA on the level of TGF-beta(1) mRNA could be markedly attenuated by pretreatment with antioxidant pyrrolidine dithiocarbamate (25micromol/L).@*CONCLUSION@#ADMA can induce the HSC activation by increasing TGF-beta(1) expression through ROS-NF-kappaB-dependent pathway. Therefore, ADMA should be a novel and endogenous activator of HSC, which may be involved in the development of liver fibrosis.
Subject(s)
Animals , Male , Rats , Actins , Arginine , Pharmacology , Cells, Cultured , Collagen Type I , Metabolism , Dose-Response Relationship, Drug , Gene Expression , Hepatocytes , Cell Biology , Metabolism , NF-kappa B , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase , Metabolism , RNA, Messenger , Genetics , Metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta , GeneticsABSTRACT
<p><b>AIM</b>To study the active constituents of Swertia davidi Franch.</p><p><b>METHODS</b>Chemical components were isolated by column chromatography and their structures were established mainly by spectroscopic means (UV, IR, NMR, 2D-NMR, MS).</p><p><b>RESULTS</b>Three substances were identified as 2,5-dimethoxyl-1, 4-dicarboxyl benzene (VIII), 1,5,8-trihydroxyl-3,4-dimethoxyl xanthone (IX) and 1,8-dihydroxyl-3-(3'-hydroxyl-butoxy) xanthone (X).</p><p><b>CONCLUSION</b>Compounds VIII and IX were isolated from Swertia davidi Franch, for the first time, whereas compound X is a new xanthone, named daviditin B with antioxygenated activity in vitro.</p>