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<p><b>OBJECTIVE</b>To optimize the culture conditions for clonal isolation of rat bone marrow-derived multipotential adult progenitor cells (rMAPC) and identify their surface markers and differentiation potentials.</p><p><b>METHODS</b>By using a low concentration of fetal bovine serum culture medium, rMAPCs were primarily isolated from bone marrow by attachment culture and clonal-like cells were selected by single cell limiting dilution. The surface antigens of the cloned rMAPC were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by lipoblasts and osteoblasts and neuroblasts differentiation induction. The expressions of Oct-4 and three embryonic germ layer markers were detected by RT-PCR.</p><p><b>RESULTS</b>Single cell-derived rMAPC could be expanded to passage 20 in vitro which still maintained active proliferation ability. The expanded rMAPCs expressed CD71, alpha-SMA and vimentin, but not CD34, CD44 and CD45. About 83% of the rMAPCs was in the resting phase(G0 + G1) of cell cycle and 17% in S + G2 + M phase. They could be induced to differentiate into adipogenic cells, osteogenic cells and neural like cells. RT-PCR demonstrated that there were expressions of oct-4 gene and three embryonic germ layer markers on the rMAPCs.</p><p><b>CONCLUSIONS</b>Cloned rMAPC can maintain the phenotypes of stem cell during in vitro culturing. It might be an potential adult stem cell source for therapeutic stem cell transplanting and tissue engineering.</p>
Subject(s)
Animals , Male , Rats , Bone Marrow Cells , Cell Biology , Cell Culture Techniques , Cell Line , Culture Media, Conditioned , Flow Cytometry , Mesenchymal Stem Cells , Cell Biology , Rats, Sprague-DawleyABSTRACT
Objective: To observe morphological changes of epithelial-mesenchymal transition in the early stage of mice renal interstitial fibrosis. Methods: Renal interstitial fibrosis was induced by unilateral ureteral obstruction(UUO) in mice. Histological and immunohistochemical methods were used to analyze pathological changes and α-SMA expression in renal tissue.Argentum hexamethylenamine staining and transmission electron microscopy were used to observe changes of the renal tubule basement membrane. Gelatin zymographic analysis was used to observe the expression of MMP2 and MMP9 in renal tissue.Results:The mice suffered from renal interstitial fibrosis were identified by histological analysis and α-SMA positive cells in renal tissue. Argentum hexamethylenamine staining and transmission electron-microscopy showed that the renal tubule basement membrane disrupted locally and renal tubule epithelial cells invaded into the renal interstitium in the early stage of renal interstitial fibrosis. Gelatin zymographic analysis showed that the expression of MMP2 and MMP9 was increased transitorily in the early stage of renal interstitial fibrosis. Conclusion: Renal tubule basement membrane disruption, renal tubule epithelial cells invasion into the renal interstitium, and the expression of MMP2 and MMP9 are involved in the development of renal interstitial fibrosis.
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Objective: To observe morphological changes of epithelial-mesenchymal transition in the early stage of mice renal interstitial fibrosis. Methods: Renal interstitial fibrosis was induced by unilateral ureteral obstruction(UUO) in mice. Histological and immunohistochemical methods were used to analyze pathological changes and α-SMA expression in renal tissue.Argentum hexamethylenamine staining and transmission electron microscopy were used to observe changes of the renal tubule basement membrane. Gelatin zymographic analysis was used to observe the expression of MMP2 and MMP9 in renal tissue.Results:The mice suffered from renal interstitial fibrosis were identified by histological analysis and α-SMA positive cells in renal tissue. Argentum hexamethylenamine staining and transmission electron-microscopy showed that the renal tubule basement membrane disrupted locally and renal tubule epithelial cells invaded into the renal interstitium in the early stage of renal interstitial fibrosis. Gelatin zymographic analysis showed that the expression of MMP2 and MMP9 was increased transitorily in the early stage of renal interstitial fibrosis. Conclusion: Renal tubule basement membrane disruption, renal tubule epithelial cells invasion into the renal interstitium, and the expression of MMP2 and MMP9 are involved in the development of renal interstitial fibrosis.
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<p><b>OBJECTIVE</b>To study the effects of Box-3 region of the leukemia inhibitory factor receptor (LIFR) alpha-chain cytoplasmic domain on the proliferation and differentiation of HL-60 cells.</p><p><b>METHODS</b>Expression vector of gp190CT3 was constructed and expressed in HL-60 cells. The expression level of gp190CT3 was assayed by immunocytochemistry. The growth of wild type and gp190CT3 transfected HL-60 cells were examined under microscope. The PCNA levels were assayed by Western blot, and the levels of CD15 by flow cytometry.</p><p><b>RESULTS</b>The gp190CT3 transfected HL-60 cells were enlarged in size and their proliferation was slower than that of wild type. The expression level of PCNA was down-regulated while the level of CD15 up-regulated in transfected HL-60 cells as compared with that of the wild type cells.</p><p><b>CONCLUSION</b>The Box-3 region of the leukemia inhibitory factor receptor alpha-chain cytoplasmic domain (gp190CT3) participates the LIFR signal transduction in inhibiting the growth and inducing the differentiation of HL-60 cells.</p>
Subject(s)
Humans , Binding Sites , Genetics , Blotting, Western , Cell Differentiation , Genetics , Physiology , Cell Proliferation , Genetic Vectors , Genetics , HL-60 Cells , Immunohistochemistry , Lewis X Antigen , Metabolism , Proliferating Cell Nuclear Antigen , Metabolism , Receptors, OSM-LIF , Genetics , Metabolism , TransfectionABSTRACT
Objective:To establish a method for isolating adipose derived stem cells(ADSCs)from resected human subcutaneous adipose tissues.Methods:ADSCs were isolated,cultured,and expanded from human subcutaneous adipose tissues.Immuno-fluorescent staining of specific molecules.FACS and multi-lineage differentiation induction were used to characterize the obtained ADSCs.Results:ADSCs obtained in this study had the characteristics of stem cells and expressed specific molecules;they also possessed a multi-lineage differentiation potential,which was genetically stable.Conclusion:ADSCs can be isolated from human subcutaneous adipose tissues,which provides a novel and abundant seeding cells for tissue engineering.