应力诱导细胞凋亡过程中活性氧与内质网应激作用机制的研究
发布时间：2019-05-28 12:15
【摘要】：目的:探讨在机械应力诱导软骨细胞凋亡的过程中,细胞内活性氧(reactive oxygen species,ROS)与内质网应激(endoplasmic reticulum stress,ERS)表达情况及两者在此作用过程中的相互作用关系方法:分离培养人膝关节软骨细胞,用应力加载系统分别将培养的细胞加载0h,6h,12h,24h和48h,分别在各受力组中加入抗氧化剂N-乙酰半胱氨酸(NAC)和半胱氨酸蛋白酶12(caspase12)特异性抑制剂Z-ATAD-FMK作为抑制剂组,应用RT-PCR检测各组细胞中凋亡相关基因Bcl-2、Bcl-2相关x蛋白(Bcl,associated X protein,Bax)及caspase12 m RNA表达量;western blot检测各组细胞中Caspase-12蛋白表达量;DCFH-DA法检测细胞中ROS的含量;流式细胞术检测细胞凋亡率。结果:RT-PCR检测6 h受力组Bax和caspase-12相对表达量增加,24 h达峰值,48 h有所回降,而Bcl-2相对表达量6 h后开始下降,12 h降到最低,24 h开始回升;ROS含量结果检测6h受力组表达量增加,24h达到峰值,48h有所回落;NAC抑制剂组ROS、Bax、caspase-12表达量均下降,Bcl-2升高。Z-ATAD-FMK抑制剂组caspase-12表达量下降,而ROS表达量不变,Western-Blot检测caspase-12蛋白表达与相应基因表达趋势相一致。凋亡检测结果与受力组caspase12表达趋势相似:6 h受力组凋亡率开始增加,24 h达峰值,48 h凋亡率有所回降;结论:机械应力可激活内质网应激途径介导软骨细胞凋亡,而且在此过程中,活性氧可能作为内质网应激上游信号分子参与其中。
[Abstract]:Objective: to investigate the intracellular reactive oxygen species (reactive oxygen species,ROS) and endoplasmic reticulum stress (endoplasmic reticulum stress, in the process of mechanical stress-induced apoptosis of cartilage cells. The expression of ERS) and the interaction between them in this process: human knee cartilage cells were isolated and cultured. The cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. the cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. Antioxidants N-acetylcystine (NAC) and Z-ATAD-FMK, a specific inhibitor of cysteinase 12 (caspase12), were added to each stress group as inhibitors. RT-PCR was used to detect apoptosis-related gene Bcl-2, in each group. Bcl-2 associated x protein (Bcl,associated X protein,Bax) and caspase12 m RNA expression; The expression of Caspase-12 protein in each group was detected by western blot, the content of ROS in cells was detected by DCFH-DA assay, and the apoptosis rate was detected by flow cytometry. Results: the relative expression of Bax and caspase-12 in the stress group increased at 6 h by RT-PCR, reached the peak at 24 h and decreased at 48 h, while the relative expression of Bcl-2 began to decrease after 6 h, reached the lowest level at 12 h, and began to rise at 24 h. The results of ROS content showed that the expression increased at 6 h, reached the peak at 24 h, and decreased at 48 h. The expression of ROS,Bax,caspase-12 in NAC inhibitor group decreased and Bcl-2 increased. In Z-ATAD-FMK inhibitor group, the expression of caspase-12 decreased, but the expression of ROS remained unchanged. The expression of caspase-12 protein detected by Western-Blot was consistent with the corresponding gene expression trend. The expression trend of caspase12 in the stress group was similar to that in the stress group: the apoptosis rate began to increase at 6 h, reached the peak at 24 h, and decreased at 48 h. Conclusion: mechanical stress can activate endoplasmic reticulum stress pathway to mediated apoptosis of chondrocytes, and reactive oxygen species may be involved as upstream signal molecules of endoplasmic reticulum stress.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3
本文编号：2487048
[Abstract]:Objective: to investigate the intracellular reactive oxygen species (reactive oxygen species,ROS) and endoplasmic reticulum stress (endoplasmic reticulum stress, in the process of mechanical stress-induced apoptosis of cartilage cells. The expression of ERS) and the interaction between them in this process: human knee cartilage cells were isolated and cultured. The cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. the cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. Antioxidants N-acetylcystine (NAC) and Z-ATAD-FMK, a specific inhibitor of cysteinase 12 (caspase12), were added to each stress group as inhibitors. RT-PCR was used to detect apoptosis-related gene Bcl-2, in each group. Bcl-2 associated x protein (Bcl,associated X protein,Bax) and caspase12 m RNA expression; The expression of Caspase-12 protein in each group was detected by western blot, the content of ROS in cells was detected by DCFH-DA assay, and the apoptosis rate was detected by flow cytometry. Results: the relative expression of Bax and caspase-12 in the stress group increased at 6 h by RT-PCR, reached the peak at 24 h and decreased at 48 h, while the relative expression of Bcl-2 began to decrease after 6 h, reached the lowest level at 12 h, and began to rise at 24 h. The results of ROS content showed that the expression increased at 6 h, reached the peak at 24 h, and decreased at 48 h. The expression of ROS,Bax,caspase-12 in NAC inhibitor group decreased and Bcl-2 increased. In Z-ATAD-FMK inhibitor group, the expression of caspase-12 decreased, but the expression of ROS remained unchanged. The expression of caspase-12 protein detected by Western-Blot was consistent with the corresponding gene expression trend. The expression trend of caspase12 in the stress group was similar to that in the stress group: the apoptosis rate began to increase at 6 h, reached the peak at 24 h, and decreased at 48 h. Conclusion: mechanical stress can activate endoplasmic reticulum stress pathway to mediated apoptosis of chondrocytes, and reactive oxygen species may be involved as upstream signal molecules of endoplasmic reticulum stress.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3
【参考文献】
相关期刊论文 前2条
1 刘兴漠;项禹诚;孙青;潘滔;黄帅;王德春;;周期性张应力对骨性关节炎软骨细胞p38 MAPK表达及其磷酸化的影响[J];中国病理生理杂志;2012年02期
2 夏元平;王立花;樊燕蓉;;细胞凋亡与内质网应激机制[J];药学与临床研究;2010年03期
，本文编号：2487048
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