周期性拉伸应力对大鼠软骨细胞代谢及凋亡的影响

Effects of cyclic tensile stress on metabolism and apoptosis of chondrocytes inrat

目的探讨周期性拉伸应力对大鼠关节软骨细胞合成、分解代谢及凋亡的影响。方法将原代大鼠软骨细胞接种在Ⅰ型胶原包被的Bioflex板中培养1d后，采用Flexcell-5000T拉伸装置在拉伸频率为0．5Hz条件下将软骨细胞拉伸24h，根据拉伸应变率的大小不同将实验分为对照组（0％拉伸）、2％拉伸组、6％拉伸组、10％拉伸组和14％拉伸组。拉伸结束后采用qPCR技术检测ColⅡ、Aggrecan、MMP-13及ADAMTS-5的mRNA表达水平；使用ELISA法检测软骨细胞上清液NO及PGE2含量；同时通过TUNEL染色及AnnexinV—FITC／PI方法检测软骨细胞凋亡。结果与对照组相比，10％和14％拉伸组ColⅡ（分别为0．738±0．11和0．58±0．13）及Aggrecan mRNA（分别为0．75±0．11和0．55±0．09）表达量降低（P〈0．05），而软骨细胞MMP-13（分别为2．47±0．47和2．88±0．36）、ADAMTS-5基因表达（2．39±0．33和2．75±0．49）mRNA表达及上清液NO（6．96±0．96和8．28±0．82）、PGE2（6．83±0．66和7．15±0．71）含量均增高（P〈0．05）；6％拉伸组的ColⅡ（1．76±0．30）及Aggrecan（1．93±0．14）mRNA表达量较对照组（1．00±0．10）增高，NO含量（3．07±0．20）较对照组（3．89±0．33）降低（P〈0．05）；2％和6％拉伸组细胞凋亡（分别为0．065±0．013，0．063±0．147）与对照组（0．045±0．008）差异无统计学意义（P分别为0．059和0．094），而10％拉伸组（0．135±0．026）和14％拉伸组（0．184±0．020）细胞凋亡显著增加（P〈0．05）。结论周期性拉伸应力对体外软骨细胞代谢及凋亡的影响具有拉伸应力大小依赖性，10％及14％拉伸应变率相应拉伸应力导致软骨细胞分解代谢及凋亡增加，6％拉伸应变率相应拉伸应力可促进软骨细胞合成代谢，而2％拉伸应变率相应拉伸应力对软骨细胞代谢和凋亡无明显影响。

Objective To investigate the effect of cyclic tensile stress （CTS） on the metabolism inand apoptosis of rat chondrocytes. Methods Primary rat chondrocytes were cultured on a Bioflex plate for one day and then stretched cyclically for 24 hours at a frequency of 0.5 Hz using a Flexcell-5000T apparatus. The cells were divided into 5 groups according to their stretching ratio： 0% （the control group）, 2%, 6%, 10% and 14%. After the stretching, Col Ⅱ,Aggrecan, MMP-13 and ADAMTS-5 mRNA were measured using qPCRs, and the NO and PGE2 levels were measured using ELISA kits. Moreover, TUNEL staining and Annexin V-FITC/PI were used to analyze the apoptosis of chondrocytes. Results Compared with the control group, the average levels of ColⅡand Aggrecan mRNA deereasedin 10% and 14% groups [（0.738±0.11） and （0.58±0.13）, （0.75±0.11） and （0.55±0.09）1. In those groups, the MMP-13 [（2.47±0.47） and （2.88±0.36）] and ADAMTS-5 mRNA level [（2.39±0.33） and （2.75±0.49）], the NO [（6.96±0.96） and （8.28±0.82）1 and PGE2level [（6.83±0.66） and （7.15±0.71）] had increased significantly. In the 6% group the average levels of Col II（1.76±0.30） and Aggrecan mRNA （1.93±0.14） of 6% group were significantly higher than the control group, but the NO level of the former （3.07±0.20） was significantly lower than the control group （3.89± 0.33）. The apoptosis rate of chondrocytesin 2% and 6% groups were （0.065±0.013） and （0.063±0.147） , without significant differences to that of the control group （0.045±0.008）.However, compared with the control group, apoptosis in the 10% and 14% groups [ （0.135±0.026） and （0.184± 0.020） ] increased significantly. Conclusion The effect of cyclic tensile stress on chondrocyte metabolism and apoptosis was magnitude-dependent. Ten percent and 14% CTS can increase the catabolism and apoptosis of chondrocytes.Ten percent and 14% strain can increase the catabolism and apoptosis of chondrocytes.Cyclic 6% strain can increase the anabolism of chondrocytes, but 2% strain has no impact on metabolism or apoptosis.