RESUMO
Objective To study the effect of dynamic stress stimulation on the expression of Sox9 mRNA and protein in metaphyseal chondrocytes in vitro, and to explore the specific mechanism of mechanical signal transduction. Methods The rat metaphyseal chondrocytes separated and cultured for the 3rd generation in vitro were randomly divided into four groups:control group (all interventions were not applied), simple dynamic pressure group (a dynamic pressure stimulus with a size of 90 mmHg and a frequency of 0.1 Hz was applied using an open pressure control culture system), simple calcium antagonist group (the concentration of 10μmol/L nifedipine was given) and dynamic pressure+calcium antagonist group (a dynamic pressure stimulus with a size of 90 mmHg, frequency of 0.1 Hz and concentration of 10 μmol/L nifedipine were given at the same time). The expression of Sox9 mRNA was detected after 24 h intervention by real-time quantitative polymerase chain reaction (RT-PCR) in four groups. The expression of Sox9 protein was detected by Western blot assay. The intracellular free Ca2+ in metaphyseal chondrocytes was labeled with Fluo-3/AM, and the average fluorescence intensity detected by laser scanning confocal scanning microscopy was compared between four groups. Results The expression of Sox9 mRNA was 3.81 times higher in dynamic stress group than that in the control group, and the protein expression level was 2.33 times higher than that of the control group (P<0.05). There were no significant differences in the expression of Sox9 mRNA and protein between the calcium antagonist group and the control group. The expressions of Sox9 mRNA and protein were lower in dynamic pressure+calcium antagonist group than those in the dynamic stress group, but which were higher than those of control group(P<0.05). The results of average fluorescence intensity showed that there was no significant difference in the intracellular free Ca2+ concentration between four groups (P > 0.05). Conclusion Dynamic stress stimulation can increase the expression of Sox9 mRNA and protein in rat metaphyseal chondrocytes. There is calcium channel involvement in the mechanical signal transduction.
RESUMO
Objective To study the effect of dynamic stress stimulation on the expression of Sox9 mRNA and protein in metaphyseal chondrocytes in vitro, and to explore the specific mechanism of mechanical signal transduction. Methods The rat metaphyseal chondrocytes separated and cultured for the 3rd generation in vitro were randomly divided into four groups:control group (all interventions were not applied), simple dynamic pressure group (a dynamic pressure stimulus with a size of 90 mmHg and a frequency of 0.1 Hz was applied using an open pressure control culture system), simple calcium antagonist group (the concentration of 10μmol/L nifedipine was given) and dynamic pressure+calcium antagonist group (a dynamic pressure stimulus with a size of 90 mmHg, frequency of 0.1 Hz and concentration of 10 μmol/L nifedipine were given at the same time). The expression of Sox9 mRNA was detected after 24 h intervention by real-time quantitative polymerase chain reaction (RT-PCR) in four groups. The expression of Sox9 protein was detected by Western blot assay. The intracellular free Ca2+ in metaphyseal chondrocytes was labeled with Fluo-3/AM, and the average fluorescence intensity detected by laser scanning confocal scanning microscopy was compared between four groups. Results The expression of Sox9 mRNA was 3.81 times higher in dynamic stress group than that in the control group, and the protein expression level was 2.33 times higher than that of the control group (P<0.05). There were no significant differences in the expression of Sox9 mRNA and protein between the calcium antagonist group and the control group. The expressions of Sox9 mRNA and protein were lower in dynamic pressure+calcium antagonist group than those in the dynamic stress group, but which were higher than those of control group(P<0.05). The results of average fluorescence intensity showed that there was no significant difference in the intracellular free Ca2+ concentration between four groups (P > 0.05). Conclusion Dynamic stress stimulation can increase the expression of Sox9 mRNA and protein in rat metaphyseal chondrocytes. There is calcium channel involvement in the mechanical signal transduction.
RESUMO
<p><b>OBJECTIVE</b>To investigate the differentiation of bone marrow mesenchymal stem cells (BMSCs) and the effects of BMSCs on the proliferation of cirrhotic fat-storing cells (CFSC) and hepatocytes in vitro.</p><p><b>METHODS</b>BMSCs and hepatocytes were isolated and harvested from the bone marrow and livers of rats. A co-culture system was set up by transwell inserts in which the two chambers were separated by a semipermeable membrane. BMSCs labeled with PKH26 were cultured with hepatocytes/CFSC in the co-culture system and also in a cell-cell direct contact culture system. Anti-albumin and anti-smooth muscle alpha-actin (alpha-SMA) antibodies were tested by using fluorescence immunocytochemistry. BMSCs and hepatocytes/CFSC cultured alone served as controls. The proliferation level of hepatocytes in the co-culture system was measured. CFSC were cultured with the conditional medium of BMSCs, and their quantities were measured microscopically.</p><p><b>RESULTS</b>Expression of albumin was observed in the hepatocytes of the two culture systems after they were cultured for 72 h but the albumin levels were higher in the cell-cell direct contact culture system (P<0.01). As compared to the controls, the number of hepatocytes was larger in the co-culture system (P<0.01). No expression of alpha-SMA in CFSC was observed in either culture system. The proliferation of CFSC was inhibited by the conditional medium of BMSCs. The longer the time of the co-culturing the more significant was the CFSC growth suppression (P<0.01).</p><p><b>CONCLUSIONS</b>BMSCs can be induced into hepatocytes by a local micro-environment formed by hepatocytes. BMSCs may promote proliferation of hepatocytes and inhibit proliferation of CFSC.</p>