不同时间周期性拉伸应力对体外培养兔软骨细胞糖胺多糖分泌的影响

Effects of cyclic tensile strain on the secretion of glycosaminoglycans in rabbit chondrocytes cultured in vitro at different times

目的观察不同时间周期性拉伸应力（CTS）对体外培养兔软骨细胞糖胺多糖（GAG）分泌的影响。方法选取2个月龄雄性新西兰大白兔9只，按照每天加载时间不同分为4 h/d、6 h/d和8 h/d组，每组3只，无菌条件下刮取双膝关节软骨，消化分离后将其接种在BioFlex 6孔培养板上，采用Flexercell-4000基底加载系统（正弦波、6%、0.5 Hz）分别加载1、2、3、4、5 d，在各个时间点分别留取上清液，Alcian blue染色沉淀法测定上清液糖胺多糖含量。结果力学加载天数不同，其上清液GAG表达也不同，差异有统计学意义（P＝0.017）。同时，随着日加载时间的延长，1、2、3 d时GAG表达量明显增加，差异有统计学意义（P＝0.012）；加载4 d、5 d时，以6 h/d组GAG表达量最高，差异有统计学意义（P＝0.023）。另外，上清液GAG含量的时间因素和处理因素之间存在交互作用（P＝0.000）。通过t检验发现，比起8 h/d组加载1 d[（10.81±0.97） g/ml]，4 h/d组加载2 d[（12.36±1.15） g/ml]GAG表达量要高，差异有统计学意义（P＝0.014）；比起6 h/d组加载2 d[（16.35±1.64） g/ml]，4 h/d组加载3 d[（16.37±1.58） g/ml]GAG表达量差异无统计学意义（P＝0.074）；比起8 h/d组加载2 d[（18.83±1.76） g/ml]，4 h/d组加载4 d[（22.92±2.10） g/ml]GAG表达量要高，差异有统计学意义（P＝0.029）；比起8 h/d组加载3 d[（26.63±2.19） g/ml]，6 h/d组加载4 d[（33.37±3.68） g/ml]GAG表达量要高，差异有统计学意义（P＝0.004）。结论周期性拉伸应变对体外软骨细胞代谢及凋亡的影响具有拉伸时间依赖性，随着拉伸时间的延长，软骨细胞外基质GAG表达量逐步增加，总加载时间在24 h时达到高峰，随后表达量开始下降。

Objective To observe the effects of cyclic tensile strain （CTS） at different times on the secretion of Glycosaminoglycans （GAG） in rabbit chondrocytes cultured in vitro.Methods Nine male New Zealand white rabbits aged two monthes were randomly divided into 4 h/d group, 6 h/d group and 8 h/d group according to the daily loading time. Three rabbits in each group were scraped with bilateral knee cartilage under aseptic condition. The original chondrocytes were inoculated on BioFlex 6-well culture plates after digestion and separation. The Flexercell-4000 substrate loading system （sine wave, 6% elongation, 0.5 Hz） was used for 1, 2, 3, 4, 5 d and supernatant was left for pecimens at these times. GAG in the supernatant was measured by Alcian blue staining method, and compare the changes of GAG secretion at different times.Results The expression of GAG in the supernatant was different with the days loaded, and the difference was statistically significant （P=0.017）. At the same time, the expression of GAG was significantly increased at 1, 2, 3 d （P=0.012）. At 4 d and 5 d, the expression of GAG was the highest at the 6 h/d team, which is statistically significant （P=0.023）. In addition, there was interaction between the time factor and the treatment factors of the supernatant （P=0.000）. Compared with the 8 h/d group 1 d loaded [（10.81±0.97） g/ml], the expression of GAG was significantly higher than that in the 4 h/d group 2 d loaded [（12.36±1.15） g/ml], and the difference was statistically significant （P=0.014）; There was no significant difference in GAG expression between the 6 h/d group 2 d loaded [（16.35±1.64） g/ml] and 4 h/d group 3 d loaded [（16.37±1.58） g/ml, P=0.074]; Compared with the 8 h/d group 2 d loaded [（18.83±1.76） g/ml], the expression of GAG was significantly higher than that in the 4 h/d group 4 d loaded [（22.92±2.10） g/ml], and the difference was statistically significant （P=0.029）; Compared with the 8 h/d group 3 d loaded [（26.63±2.19） g/ml], the expression of GAG was significantly higher than that in the 6 h/d group 4 d loaded [（33.37±3.68） g/ml], and the difference was statistically significant （P=0.004）.Conclusion The effect of CTS on the metabolism and apoptosis of chondrocytes in vitro was time-dependent. The expression of GAG in the extracellular matrix increased gradually with the prolongation of the stretching time, and the total loading time reached the peak at 24 h, and then the expression began to decline.