动态压应力促进软骨前体干细胞Sox9和细胞外基质表达的实验研究

Dynamic compressive pressure promotes expression of Sox9 and extracellular matrix of precartilaginous stem cells：an experimental study

目的：研究间断性动态压应力对体外培养的大鼠软骨前体干细胞Sox9及软骨特异性细胞外基质表达的影响。方法细胞免疫磁珠分选并体外培养大鼠软骨前体干细胞（PSC），使用自行设计制备的细胞压力装置对细胞进行间断性（1 d刺激12 h，分别1 d，3 d和7 d）的压应力刺激（90 mmHg，1 mmHg=0.133 kPa），未刺激组为对照组，采用荧光实时定量聚合酶链反应检测各组细胞的Sox9和软骨特异性细胞外基质（Ⅱ型胶原）的基因表达情况，Western印迹检测细胞Sox9和Ⅱ型胶原蛋白表达水平并进行分析。结果在动态压应力刺激下，软骨前体干细胞各时间点Sox9和Ⅱ型胶原的基因表达水平明显增加并高于对照组（P〈0.05）；压力刺激1 d，3 d和7 d后，于对照组相比，其Sox9和Ⅱ型胶原mRNA表达水平分别增加了约1.9倍和2.1倍，3.5倍和3.4倍以及4.6倍和4.3倍。1 d压力组Sox9和Ⅱ型胶原蛋白表达与对照组无明显差异（P〉0.05），3 d和7 d时间点的蛋白表达增加并高于对照组（P〈0.05）；Sox9和Ⅱ型胶原基因和蛋白表达水平都随着压应力刺激时间增加而增加。结论间断性动态压应力可以明显增加大鼠软骨前体干细胞Sox9和Ⅱ型胶原的表达，并可能会促进软骨前体干细胞向软骨细胞的分化。

Objective To investigate the effect of intermittent dynamic compressive pressure on the expression of Sox9 and specific extracellular matrix of in vitro cultured precartilaginous stem cells（PSCs）in rats. Methods The rat PSCs were isolated by magnetic activated cell sorting and cultured in vitro. Using self-designed and prepared cell pressure device to intermittently stimulate（12 h at 1 d,3 d and 7 d）the cells with compressive pressure（90 mmHg,1 mmHg=0.133 kPa）. The unstimulated group was included as the control group. The gene expression of Sox9 and cartilage-specific extracellular matrix（typeⅡcollagen） in each group were measured by real-time quantitative polymerase chain reaction （qPCR）. The expression levels of Sox9 and type Ⅱ collagen protein were determined by Western blotting. Results Under the dynamic compressive pressure,the expression levels of Sox9 and typeⅡcollagen of PSC were significantly increased compared with those in the control groups at each time point（P〈0.05）. At 1 d,3 d and 7 d after the stimulation with compressive pressure,compared with the control group,the mRNA expression levels of Sox9 and typeⅡcollagen were increased by approximately 1.9 times and 2.1 times,3.5 times and 3.4 times,as well as 4.6 times and 4.3 times,respectively. There were no significant differences in the expression levels of Sox9 and type Ⅱ collagen protein at 1 d between the compressive pressure group and the control group （P〉0.05）. The protein expression levels of Sox9 and type Ⅱ collagen were increased at 3 d and 7 d in the compressive pressure group,which were higher than those in the control group（P〈0.05）. The gene and protein expression levels of Sox9 and typeⅡcollagen increased along with the increasing time of stimulation. Conclusion The intermittent dynamic compressive pressure can significantly increase the expression of Sox9 and typeⅡcollagen in rat PSC,which may promote the differentiation of PSC into chondrocytes.