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Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry ( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio|≥1. 5, P< 0. 05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up-or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.
ABSTRACT
Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry ( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio|≥1. 5, P< 0. 05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up-or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.
ABSTRACT
This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor beta(1) (TGF-beta(1)), insulin-like growth factor I (IGF-I) and vitamin C (V(C)), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.
Subject(s)
Humans , Bone Marrow Cells , Cell Biology , Cell Culture Techniques , Methods , Cell Differentiation , Cells, Cultured , Cryopreservation , Methods , Mesenchymal Stem Cells , Cell Biology , Pluripotent Stem Cells , Cell Biology , Tissue Engineering , MethodsABSTRACT
<p><b>OBJECTIVE</b>To explore the biological characteristics of mesenchymal stem cells (MSC) derived from umbilical cord blood (UCB) and their supporting capacities in ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs).</p><p><b>METHODS</b>Low-density mononuclear cells (MNCs) from UCB were cultured in IMDM containing 20% FBS to form confluent adherent cells through 15 passages. Some cytokines in the conditioned medium were determined with ELISA. UCB-derived adherent cells were displayed with antibodies and analyzed with flow cytometry. The supporting capacity of UCB-derived adherent cells for ex vivo expansion of CD34(+) cells was assayed by co-culture in a two step culture. UCB-derived adherent cells were induced for chondrogenic differentiation with chondrogenic medium, and the induced cells were analyzed for the type II pro-collagen gene expression with RT-PCR.</p><p><b>RESULTS</b>The mean number of adherent fibroblast like colonies derived from UCB was (3.5 +/- 0.7)/10(6) MNCs. UCB-derived MSCs could survive for at least 15 passages of expansion. In their undifferentiated status, UCB-derived MSCs were CD13(+), CD29(+), CD90(+), CD105(+), CD166(+), SH2(+), SH3(+), SH4(+), CD45(-), CD34(-), and CD14(-). Stem cell factor (SCF), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) could be detected in the supernatant of the cultures. The MSCs cultured in chondrogenic media could differentiate into chondrogenic cells and express type II pro-collagen mRNA. UCB-derived MSCs could support the proliferation and differentiation of UCB CD34(+) cells in vitro.</p><p><b>CONCLUSION</b>UCB-derived MSCs are similar to those derived from adult bone marrow and can support the proliferation of hematopoietic stem/progenitor cells.</p>
Subject(s)
Humans , Antigens, CD34 , Metabolism , Cell Adhesion , Cell Differentiation , Cell Proliferation , Cells, Cultured , Coculture Techniques , Collagen Type II , Genetics , Cytokines , Metabolism , Enzyme-Linked Immunosorbent Assay , Fetal Blood , Cell Biology , Gene Expression , Hematopoietic Stem Cells , Cell Biology , Metabolism , Mesenchymal Stem Cells , Cell Biology , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Bone marrow mesenchymal stem cells (MSCs) are defined as pluripotent cells which have high self-renewal capacity and multipotentiality for differentiation. Because of their characteristics of supporting hematopoietisis, multipotentiality for differentiation and their possible use for both cell and gene engineerings, MSCs will have important value in clinic use.
Subject(s)
Animals , Humans , Cell Differentiation , Genetics , Physiology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Cell Biology , Metabolism , Physiology , Models, BiologicalABSTRACT
<p><b>OBJECTIVE</b>To explore the rapid neutrophil engraftment and long-term hematopoietic reconstitution.</p><p><b>METHODS</b>Mononuclear cells (MNCs) were isolated from 5-Fu treated male BDF1 mouse bone marrow and CD(34)(+)/c-kit(+) cells were selected from the MNCs by using MoFlo Cell Sorter. MNCs and CD(34)(+)/c-kit(+) cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) in a two-step expansion. The expanded cells were then transplanted into sublethally irradiated female BDF1 mice.</p><p><b>RESULTS</b>Co-culture with MSCs resulted in 10.8-, 4.8-, 65.9- and 38.8-fold increases yields of median total nucleated cells, CD(34)(+) cells, GM-CFC and HPP-CFC, respectively, as for the MNCs culture, and 76.1-, 2.9-, 71.7- and 51.8-fold increase respectively for the CD(34)(+)/c-kit(+) cell culture. The expanded cells could rapidly engraft in the sublethally, irradiated mice, reconstitute their hematopoiesis, and be detected in the recipients bone marrow 2 months after transplantation.</p><p><b>CONCLUSIONS</b>Hematopoietic stem/progenitor cells co-cultures with MSCs in two-step expansion could increase expansion yields of total nucleated cells, GM-CFC and HPP-CFC. The availability of increased numbers of expanded cells may result in more rapid engraftment of neutrophils following infusion to transplant recipients.</p>