CAJ | 학술논문

目的观察在周期性应力介导成肌细胞凋亡中内质网应激需肌醇酶-1(IRE-1)-c-jun氨基端激酶(JNK)信号通路的作用.方法以大鼠L6成肌细胞为实验对象,构建体外培养-力学刺激模型.应用应力加载装置分别加1、2、6、12、24 h的周期性张应力(拉伸变形率为15％,频率为10次循环/分),对照组不加力(0 h).Hochest 33258染色、膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)/碘化丙锭(PI)流式细胞术分别检测凋亡细胞的数目及凋亡率;反转录-聚合酶链反应(RT-PCR)检测内质网应激相关因子C/EBP同源蛋白(CHOP)、凋亡信号调节激酶-1(ASK-1)、肿瘤坏死因子受体相关因子2(TRAF2)和JNK mRNA的表达变化;加入IRE-1特异性抑制剂STF-083010,检测ASK-1、TRAF2和JNK mRNA的表达变化.结果随应力加载时间的延长,凋亡细胞的数目、凋亡率呈一致上升趋势,且在24h达峰值[(14.34±0.77)个];CHOP、ASK-1、TRAF2mRNA表达量随着应力加载时间的延长逐渐上升,并在24 h达最高值,分别为4.19±1.76、3.46±0.84、3.78±1.38(P ＜0.05),JNK mRNA的表达量在0～2h逐渐上升后逐渐下降,随后又逐渐上升在24 h达最高值(3.04±0.79,P＜0.05);加入大鼠IRE-1特异性抑制剂STF-083010后,TRAF2、ASK-1表达量均明显减少(P＜0.05).结论周期性张应力可诱导成肌细胞的凋亡,凋亡率随着应力加载时间的延长而升高.CHOP、TRAF2、ASK-1、JNK mRNA的表达量升高,提示内质网相关的凋亡途径参与了应力介导的成肌细胞的凋亡.加入IRE-1抑制剂后,TRAF2、ASK-1、JNK mRNA表达量均明显降低,提示IRE-1-JNK通路参与了应力介导的成肌细胞凋亡.
목적 관찰재주기성응력개도성기세포조망중내질망응격수기순매-1(IRE-1)-c-jun안기단격매(JNK)신호통로적작용.방법 이대서L6성기세포위실험대상,구건체외배양-역학자격모형.응용응력가재장치분별가1、2、6、12、24 h적주기성장응력(랍신변형솔위15％,빈솔위10차순배/분),대조조불가력(0 h).Hochest 33258염색、막련단백V(Annexin V)-이류청산형광소(FITC)/전화병정(PI)류식세포술분별검측조망세포적수목급조망솔;반전록-취합매련반응(RT-PCR)검측내질망응격상관인자C/EBP동원단백(CHOP)、조망신호조절격매-1(ASK-1)、종류배사인자수체상관인자2(TRAF2)화JNK mRNA적표체변화;가입IRE-1특이성억제제STF-083010,검측ASK-1、TRAF2화JNK mRNA적표체변화.결과 수응력가재시간적연장,조망세포적수목、조망솔정일치상승추세,차재24h체봉치[(14.34±0.77)개];CHOP、ASK-1、TRAF2mRNA표체량수착응력가재시간적연장축점상승,병재24 h체최고치,분별위4.19±1.76、3.46±0.84、3.78±1.38(P ＜0.05),JNK mRNA적표체량재0～2h축점상승후축점하강,수후우축점상승재24 h체최고치(3.04±0.79,P＜0.05);가입대서IRE-1특이성억제제STF-083010후,TRAF2、ASK-1표체량균명현감소(P＜0.05).결론 주기성장응력가유도성기세포적조망,조망솔수착응력가재시간적연장이승고.CHOP、TRAF2、ASK-1、JNK mRNA적표체량승고,제시내질망상관적조망도경삼여료응력개도적성기세포적조망.가입IRE-1억제제후,TRAF2、ASK-1、JNK mRNA표체량균명현강저,제시IRE-1-JNK통로삼여료응력개도적성기세포조망.
Objective To explore the role of inositol requiring enzyme-1 (IRE-1)-c-Jun N-terminal kinase (JNK) signaling pathways in stretch-induced apoptosis of myoblasts.Methods As experimental objects,L6 rat myoblasts were used to construct the culture-mechanical stimulation model in vitro.Then cells were subjected to 15％ surface elongation with a frequency of 10 cycles/min cyclic stretch using multi-channel stress loading system.Cells were harvested after different durations (1,2,6,12,24 h) of cyclic exposure.After cyclic stretch,Hochest 33258 staining and Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) staining was performed to observe the morphology of the apoptotic cells.Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of endoplasmic reticulum stress related factors [C/EBP homologous protein (CHOP),tumor necrosis factor receptor associated factor2 (TRAF2),apoptosis signal regulating kinase-1 (ASK-1) and JNK].IRE-1 specific inhibitor (STF-083010) was used to detect the TRAF2,ASK-1 and JNK mRNA expression to investigate the role of IRE-1-JNK signaling pathway in stretch-induced apoptosis.Results The stretch-induced apoptosis rate of myoblasts was increased in a time-dependent manner,peaked at 24 h (14.34 ± 0.77).CHOP,TRAF2 and ASK-1 mRNA expression levels were increased with the extension of stretch time,and reached the maximum at 24 h [(4.19 ± 1.76),(3.46 ±0.84),and (3.78 ± 1.38),P ＜0.05];but JNK mRNA expression was decreased followed by an increase range from 0 h to 2 h,and then increased again within 24 h and peaked at 24 h (3.04 ± 0.79,P ＜ 0.05).After treatment with IRE-1 specific inhibitor (STF-083010),the real-time PCR results showed that TRAF2 and ASK-1 mRNA expression was significantly decreased (P ＜ 0.05).Conclusion Cyclic stretch can induce myoblast apoptosis and within 24 h the apoptosis rate of myoblasts was increased in a time-dependent manner.The increase of CHOP,TRAF2,ASK-1,and JNK mRNA indicates that endoplasmic reticulum stress may involve in stretch-induced apoptosis.After treatment with IRE-1 specific inhibitor,the significant decrease of CHOP,TRAF2,ASK-1 and JNK mRNA,and JNK protein indicates that IRE-1-JNK signaling pathway may involve in stretch-induced apoptosis.