过度周期性机械应力刺激可引起软骨细胞炎症反应及凋亡  

Effect of periodic mechanical stress on inflammatory responses and apoptosis of chondrocytes

作　　者：伍伟挺 黎润光[2] 曹生鲁 梁双武[2] Wu Weiting;Li Runguang;Cao Shenglu;Liang Shuangwu(Xintang Hospital of Zengcheng District,Guangzhou 511340,Guangdong Province,China;Nanfang Hospital,Southern Medical University,Guangzhou 511340,Guangdong Province,China)

机构地区：[1]广州市增城区新塘医院,广东省广州市511340 [2]南方医科大学南方医院,广东省广州市511340

出　　处：《中国组织工程研究》2021年第29期4608-4613,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金项目(2014A030313338),课题名称:体外软骨细胞高应力损伤模型的构建及损伤机制的研究,项目负责人:黎润光。

摘　　要：背景:软骨细胞为软骨的惟一细胞成分,软骨细胞表型丢失是骨关节炎的特征性改变,而周期性机械应力刺激对软骨细胞的生物特性有重要影响,明确周期性机械应力对软骨细胞的作用机制可为骨关节炎的治疗提供借鉴。目的:观察周期性机械应力对体外软骨细胞炎症反应和凋亡的影响。方法:将原代大鼠软骨细胞接种于Ⅰ型胶原包被的Bioflex板中培养1 d后,采用细胞体外加力实验系统(Flexcell-5000T Tension System)处理细胞,在拉伸频率为0.5 Hz条件下将软骨细胞拉伸24 h,依据拉伸应变率大小分为对照组(0%拉伸)、2%拉伸组、6%拉伸组、8%拉伸组、10%拉伸组、14%拉伸组。拉伸结束后采用ELISA法检测软骨细胞炎症反应(白细胞介素1β、肿瘤坏死因子α),TUNEL染色及Annexin V-FITC/PI法检测凋亡情况,利用RT-PCR检测内质网应激信号分子(半胱氨酸蛋白酶12、蛋白激酶R样内质网激酶、葡萄糖调节蛋白78)。结果与结论:①光镜下可观察到正常细胞(对照组)为多角形,形态不规则;而随机械拉伸应变率增加,细胞有不同程度脱落,边缘模糊变圆,出现凋亡现象;②与对照组相比,施力组白细胞介素1β、肿瘤坏死因子α升高,尤其是10%拉伸组、14%拉伸组升高较明显(P<0.05);③周期性机械应力作用下,2%、6%拉伸组细胞凋亡率分别为(12.45±1.64)%,(12.48±1.75)%,与对照组(12.14±1.43)%相比差异无显著性意义(P>0.05);而8%、10%、14%拉伸组软骨细胞凋亡率[(29.18±3.04)%,(34.12±3.57)%,(44.32±45.08)%]显著增加(P<0.05);④RT-PCR检测结果显示,8%、10%、14%拉伸组半胱氨酸蛋白酶12、蛋白激酶R样内质网激酶、葡萄糖调节蛋白78的mRNA表达量明显高于对照组(P<0.05);⑤提示周期性机械应力可加速软骨细胞凋亡过程,促进软骨细胞炎症反应,可能与内质网应激通路启动有关。BACKGROUND:As chondrocyte is the only cell component of cartilage,chondrocyte phenotype loss is a characteristic change of osteoarthritis,while periodic mechanical stress stimulation has great influence on the biological characteristics of chondrocytes.Therefore,clarification of the mechanism of periodic mechanical stress on chondrocytes can provide reference for treating osteoarthritis.OBJECTIVE:To observe the effect of periodic mechanical stress on inflammatory responses and apoptosis of chondrocytes in vitro.METHODS:Primary chondrocytes from rats were inoculated into a Bioflex plate coated with type I collagen and were cultured for 1 day.Then,the cells were treated with Flexcell-5000T Tension System.Under the stretching frequency of 0.5 Hz,the chondrocytes were stretched for 24 hours.According to the stretching strain rate,the chondrocytes were divided into control group(0%stretching),2%stretching group,6%stretching group,8%stretching group,10%stretching group and 14%stretching group.Inflammatory responses of chondrocytes(interleukin-1β,tumor necrosis factor alpha)were detected by ELISA,cell apoptosis was detected by TUNEL staining and Annexin V-FITC/PI method,and the expression of endoplasmic reticulum stress signal molecules[Caspase-12,PKR-like endoplasmic reticulum kinase(PERK),glucose regulated protein 78(GRP78)]was detected using RT-PCR.RESULTS AND CONCLUSION:Under normal light microscope,normal cells(control group)were polygonal and irregular in shape.With the increase of mechanical stretching strain rate,the cells were exfoliated to varying degrees,with the edge being blurred and rounded.Moreover,cell apoptosis appeared.Compared with the control group,interleukin-1βand tumor necrosis factor alpha levels were higher in the force groups,especially in the 10%and 14%stretching groups(P<0.05).Under the action of periodic mechanical stress,the apoptosis rates of the 2%stretching group and 6%stretching group were(12.45±1.64)%and(12.48±1.75)%,respectively,which did not significantly differ from that of the

关 键 词：周期性机械应力 体外研究 软骨细胞 炎症反应 凋亡 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]