Effects of aldosterone on osteoblast proliferation, differentiation and osteogenic gene expressions in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of aldosterone on cell proliferation, alkaline phosphatase (AKP) activity and osteogenic gene expression in rat osteoblasts and explore the mechanisms.</p><p><b>METHODS</b>Osteoblasts isolated from the skull of neonatal SD rats by enzyme digestion were cultured and treated with different concentrations of aldosterone. The cell proliferation and AKP activity were evaluated using CCK-8 assay kit and AKP assay kit, respectively. The effects of aldosterone on mRNA and protein expressions of the osteogenic genes and epithelial sodium channel (ENaC) gene were investigated using semi-quantitative PCR and Western blotting.</p><p><b>RESULTS</b>Compared with the control cells, the cells treated with 0.01-1.0 µmol/L aldosterone showed obviously enhanced proliferation while lower (1×10µmol/L) or higher (10 µmol/L) concentrations of aldosterone did not significantly affect the cell proliferation. Aldosterone within the concentration range of 1×10to 10 µmol/L did not cause significant changes in AKP activity in the osteoblasts. Treatment with 0.01 to 1.0 µmol/L aldosterone significantly upregulated the expressions of the osteogenic genes and α-ENaC gene at both the mRNA and protein levels.</p><p><b>CONCLUSION</b>Aldosterone within the concentration range of 0.01-1.0 µmol/L stimulates the proliferation and osteogenic gene expressions and enhances α-ENaC gene expression in rat osteoblasts in vitro, suggesting the possibility that ENaC participates in aldosterone-mediated regulation of osteoblast functions.</p>

Role of epithelial sodium channel in rat osteoclast differentiation and bone resorption / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts.</p><p><b>METHODS</b>Multinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts.</p><p><b>RESULTS</b>s Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K.</p><p><b>CONCLUSION</b>s ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.</p>

Establishment of a tight tetracycline-controlled HCV-C double transgenic mouse model / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a tight tetracycline-controlled HCV-C double transgenic mouse model.</p><p><b>METHODS</b>By crossbreeding of ApoE-rtTA-tTS transgenic mice with TRE-HCV-C transgenic mice, the double transgenic mice were produced in the F1 generation. The presence of HCV-C and tTS gene in the F1 generation was confirmed by PCR, followed by further identification and quantification of the transgene using Southern blot hybridization. The expression of HCV-C in the liver of the mouse model was detected immunohistochemically.</p><p><b>RESULTS AND CONCLUSION</b>Two transgenic mice were obtained, which contained ApoE-rtTA-tTS and TRE-HCV-C genes in the genome. Five founders contained HCV-C gene as confirmed by PCR and Southern blot hybridization. The tight tetracycline-controlled system may facilitate further study of HCV-C gene expression and gene therapy of hepatic cellular carcinoma.</p>