不同加载时间周期性张应力对大鼠颅底软骨细胞的影响

Effect of cyclic tensile stress on expression of type Ⅱ collagen and Sox9 in rat cranial base synchondrosis

目的 :构建颅底软骨细胞体外培养力学刺激模型,研究周期性张应力对颅底软骨细胞主要细胞外基质(Ⅱ型胶原)和Sox9表达的影响。方法:采用FX-5000T应力加载系统对体外培养的第2代大鼠颅底软骨细胞分别施加3、6、12、24 h的周期性张应力,加力值均为10%形变率,频率均为1 Hz。加力后即刻收集细胞,提取总RNA,实时荧光定量PCR技术检测颅底软骨细胞Ⅱ型胶原(type-Ⅱcollagen,Col-Ⅱ)和Sox9 m RNA的表达。采用SPSS 17.0软件包对数据进行统计学分析。结果:与对照组(加力0 h)相比,加力3 h组Col-Ⅱ和Sox9表达下降,且后者有显著差异(P<0.05);加力6 h组Col-Ⅱ和Sox9表达显著下降(Col-Ⅱ,P<0.01;Sox9,P<0.05);加力12 h组Col-Ⅱ和Sox9表达较6 h组有所上升,与对照组相比差异无统计学意义;加力24 h组中,两者表达与对照组相比显著升高(P<0.05)。结论:周期性张应力可影响颅底软骨细胞增殖及细胞外基质合成,加力短时间基质合成抑制,增加加力时间则明显促进基质合成,且成软骨分化标志物Sox9 m RNA表达水平差异先于Col-Ⅱ出现。

PURPOSE： To establish an in vitro mechanical stimulation model of cranial base synchondrosis chondrocytes,and to study the effect of cyclic tensile stress on the main extracellular matrix of rat cranial base synchondrosis.METHODS： Cyclic tensile stress was imposed to the second passage of cranial base synchondrosis chondrocytes for 3, 6,12 and 24 hours respectively by using a Flexcell Strain Unit-5000 T（10% surface elongation, 1 Hz）. After mechanical loading, the total RNA of the cells harvested from six-well Bio Flex was extracted. Real-time quantitative RT-PCR was performed to quantify the m RNA levels of type Ⅱ collagen and Sox9. The data were analyzed with SPSS 17.0 software package. RESULTS： Compared with the control group（0 h group）, the m RNA expression of type Ⅱ collagen was decreased after 3 hours of loading, but not statistically significant; While the expression of Sox9 decreased significantly（ P〈0.05）. In the 6 h group, the expression of Col-Ⅱ and Sox9 decreased significantly（P〈0.01 and 0.05, respectively）. The expression of Col-Ⅱ and Sox9 increased in the 12 h group. The 24 h group showed significant increase in both type Ⅱ collagen and Sox9（P〈0.05）. CONCLUSIONS： The results illustrate that cyclic tensile stress can affect the synthesis of the main extracellular matrix of cranial base synchondrosis in vitro. Expression of type Ⅱ collagen and Sox9 can be inhibited during early stage of mechanical loading. However, when loading time extends, the mechanical stimuli greatly promotes the expression of type Ⅱ collagen and Sox9. The reaction of Sox9 in this in vitro mechanical stimulation model happens earlier than that of type Ⅱ collagen.