动态压力对大鼠干骺端软骨细胞Sox9 mRNA及蛋白表达水平的影响

摘要

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.%目的 研究动态压力刺激对体外培养的大鼠干骺端软骨细胞Sox9 mRNA及蛋白表达水平的影响,探讨动态压力信号转导的可能机制.方法 体外分离培养的大鼠干骺端软骨细胞培养至第3代时随机分为4组:对照组,所有干预措施均不施加;单纯动态压力组,采用开放式压力控制培养系统施加大小为90 mmHg、频率为0.1 Hz的动态压力刺激;单纯钙离子拮抗剂组,施加浓度为10μmol/L硝苯地平干预;动态压力+钙离子拮抗剂组,同时进行90 mmHg、0.1 Hz的动态压力刺激和浓度为10μmol/L硝苯地平干预.干预24 h后进行实时定量PCR检测各组细胞Sox9 mRNA表达情况,Western blot检测各组细胞Sox9蛋白表达情况.以Fluo-3/AM标记干骺端软骨细胞内游离Ca2+,激光共聚焦扫描显微镜扫描并比较各组细胞平均荧光强度.结果 实时定量PCR结果显示,单纯动态压力组细胞内Sox9 mRNA平均表达水平是对照组细胞的3.81倍,蛋白表达水平是对照组细胞的2.33倍(P<0.05);单纯钙离子拮抗剂组细胞Sox9 mRNA表达量和蛋白表达量与对照组差异无统计学意义;动态压力+钙离子拮抗剂组细胞Sox9 mRNA表达量和蛋白表达量均较单纯动态压力组低,但均较对照组升高(P<0.05).各组细胞平均荧光强度结果表明各组细胞内游离Ca2+浓度差异无统计学意义(P>0.05).结论 动态压力刺激可以增加大鼠干骺端软骨细胞Sox9 mRNA及蛋白的表达,且在该力学信号转导中有钙离子通道的参与.