中华临床医师杂志(电子版) 2017年2月,11卷4期

基础论著

活性氧激活内质网诱导软骨细胞凋亡的研究

刘超1 夏长所1 李晓飞1 金丹凤2 吕成昱1

266003 山东青岛,青岛大学医学院附属医院关节外科1;266000 山东青岛,青岛市海慈医疗集团儿二科2
吕成昱,Email: lvchengyu@medmail.com.cn

摘要:目的 探讨应力诱导的人软骨细胞凋亡过程中活性氧(ROS)与内质网应激(ERS)的作用关系。方法 分离培养人膝关节软骨细胞,应用细胞牵张力加载系统对各组软骨细胞分别加载0 h、6 h、12 h和24 h,在各受力组中加入抗氧化剂N-乙酰半胱氨酸(NAC)作为抑制剂组。RT-PCR和Western blot检测各组细胞中内质网应激标志因子Caspase-12的表达;DCFH-DA法检测细胞中ROS的含量;流式细胞术检测细胞凋亡率,SPSS 17.0统计软件处理实验数据,组间比较采用t检验。结果 RT-PCR和Western blot检测Caspase-12结果显示:2 h表达量开始增加;24 h达峰值;NAC抑制剂组,与受力组相比,表达明显降低(P<0.05);DCFH-DA检测ROS结果显示:受力2 h后含量明显增加,24 h达峰值;NAC抑制剂组,相较于受力组,明显降低(P<0.05);细胞凋亡率检测结果显示:受力2 h后凋亡率开始增加,24 h达峰值,而NAC抑制剂组,与受力组相比,显著下调(P<0.01)。结论 应力诱导软骨细胞凋亡过程中,ROS可能作为上级信号激活内质网应激途径参与软骨细胞凋亡。

关键词:周期性张应力; 内质网应激; 活性氧; 软骨细胞; 半胱氨酸蛋白酶12

国家自然科学基金(81601875)

Stretch induced apoptosis of chondrocytes via the pathway of reactive oxygen species activated endoplasmic reticulum stress

Liu Chao1, Xia Changsuo1, Li Xiaofei1, Jin

Danfeng2, Lyu Chengyu1. 1Department of Join Surgery, the Affiliated Hospital of Qingdao University, Qingdao 266003, China; 2Department of Paediatrics, the Haici Hospital, Qingdao 266000, China
Lyu Chenyu, Email: lvchengyu@medmail.com.cn[DOI:10.3877/cma.j.issn.1674-0785.2017.04.013

Abstract:Objective To investigate the relationship between reactive oxygen species (ROS) and endoplasmic reticulum stress (ERS) in the process of stretch induced apoptosis of chondrocytes. Methods The human articular chondrocytes was cultured and divided into groups that disposed with stressing 0 h, 6 h, 12 h, 24 h, 48 h, respectively. And the groups disposed with stretch and antioxidant: N-acetyl cysteine as inhibitor group. The expressions of the Caspase-12 were assessed by revers transcription-polymerase chain reaction (RT-PCR) and Western blot. The intracellular ROS were detected with DCFH-DA. Apoptosis was evaluated by flow cytometry. The experimental data were handled with the software of SPSS 17.0. Results The result of the RT-PCR and Western blot had shown that the expression of the Caspase-12 began to increase at 2 h, and reached highest at 24 h; The Caspase-12 in the NAC group was decreasing than stretch group (P<0.05); The expression of the ROS began to increase at 2 h reached the highest at 24 h, however inhibitor group of the ROS was decreased when compared with the stretch group (P<0.05). The result of the cell apoptotic in the stretch group was increased at 2 h and reached highest at 24 h, however, compared with the stretch group, the cell apoptotic in the inhibitor group was lower obviously (P<0.01). Conclusion The ROS may be the superior signal that activated ERS in the process of apoptosis of chondrocytes which was induced by stretch.

Keywords: Cyclic stress; Endolplasmic reticulum stress; Reactive oxygen pecies; Chordroytes; Caspase-12

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(编辑:戚红丹 收稿日期:2016-10-31)