摘要
目的:研究周期性张应力对L6大鼠成肌细胞凋亡的影响,探讨蛋白激酶R样内质网激酶(protein kinase Rlike ER kinase,PERK)在这一过程中的作用及机制。方法:构建L6细胞力学刺激模型,加载参数为细胞拉伸形变率15%,频率10个循环/min,每循环包括3 s拉伸及3 s舒张,加力时间为2、6、12、24 h,以不加力组为对照组。应用DAPI染色观察各组细胞凋亡情况;应用实时荧光定量PCR检测各组PERK、CHOP mRNA的表达;应用Western免疫印迹检测各组PERK、p-PERK的表达变化。加入PERK抑制剂后重复实验,检测各组细胞凋亡情况,PERK、CHOP mRNA的表达变化及p-PERK的蛋白表达变化。采用SPSS17.0软件包进行统计学分析。结果:DAPI染色显示,L6大鼠成肌细胞在周期性应力诱导下发生凋亡,24 h时达到凋亡最大值。PERK被抑制后,加力组的细胞凋亡程度与对照组无显著差别。RT-PCR结果显示,随着加力时间的延长,CHOP mRNA的表达逐渐增加;PERK mRNA表达无差异。Western免疫印迹结果显示,p-PERK蛋白表达水平随加力时间延长而逐渐升高,总蛋白PERK的表达无明显变化;加入PERK抑制剂后,CHOP的mRNA表达与对照组无显著差异,p-PERK蛋白表达与对照组无显著差异。结论:内质网应激关键因子PERK参与了周期性应力条件下的成肌细胞凋亡,其作用机制可能与PERK磷酸化有关。
PURPOSE:This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved.METHODS:L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully.The myoblasts were imposed tension for0,2,6,12 and 24 hours respectively by multi-channel cell stress loading system.The force value was 15% cell deformation and the frequency was 10 cycles/min.Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds,and the group without tension was used as the control group.The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate;the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot.PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch.SPSS 17.0 software package was used to analyze the data statistically.RESULTS:DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast.Apoptosis rate showed a trend of rising gradually over time,peaked at 24 h.After dealt with the inhibitor of PERK,the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control.The results of real-time PCR showed that the m RNA of CHOP was increased with the extension loading time,while the mRNA of PERK showed no difference compared with the control.Western blot results showed that the protein level of p-PERK was increased with the extension of loading time,while the expression of PERK showed no difference compared with the control group.When PERK inhibitor added,the m RNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control.CONCLUSIO
作者
嵇国平
田一弘
刘美希
沈莹
沈刚
JI Guo-ping;TIAN Yi-hong;LIU Mei-xi;SHEN Ying;SHEN Gang(Shanghai Hengzheng Dental Clinic. Shanghai 200040;Department of Orthodontics,School and Hospital of Stomatology,Qingdao University. Qingdao 266555,Shandong Province;Shanghai ByBo Dental Hospital,United Orthodontic Institutions,ByBo Dental Group. Shanghai 200001,China)
出处
《上海口腔医学》
CAS
CSCD
北大核心
2019年第3期259-263,共5页
Shanghai Journal of Stomatology
