CAJ | 학술논문

背景:在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、实验条件的不易控制而很难得到满意结果.目的:在成功构建成肌细胞体外培养-力学刺激模型的基础上,研究p38MAPK信号通路在成肌细胞凋亡中的作用及其机制.方法:将体外培养的C2C12细胞分为对照组和SB203580组,SB203580组中加入 20 mmol/L的p38MAPK抑制剂SB203580.应用细胞应力加载装置Flecell Strain Unit-5000T给细胞提供15%的力值,分别施加0,6,12,24 h的周期性张应力.每分钟10个循环,每循环包括3 s牵张,3 s松弛.Hoechst 33258染色观察细胞的形态学变化;流式细胞仪检测细胞凋亡情况;RT-PCR法检测促凋亡基因bax mRNA的表达;Western blot检测信号通路中p38MAPK和p-p38MAPK蛋白的表达.结果与结论:随着加力时间的延长,细胞逐渐出现核固缩及凋亡小体,凋亡率增加(P < 0.05),bax mRNA表达增多(P < 0.05);细胞p38MAPK和p-p38MAPK蛋白均在加力6 h达到最低,此后逐渐升高.p38MAPK抑制剂SB203580可抑制加力引起的细胞凋亡,减少bax mRNA及p38MAPK和p-p38MAPK蛋白的表达(P < 0.05).说明p38MAPK信号通路在应力介导的成肌细胞凋亡中起到重要的作用.
배경:재체내조건하,세포역학적공능연구인기소처생리배경적복잡성、실험조건적불역공제이흔난득도만의결과.목적:재성공구건성기세포체외배양-역학자격모형적기출상,연구p38MAPK신호통로재성기세포조망중적작용급기궤제.방법:장체외배양적C2C12세포분위대조조화SB203580조,SB203580조중가입 20 mmol/L적p38MAPK억제제SB203580.응용세포응력가재장치Flecell Strain Unit-5000T급세포제공15%적력치,분별시가0,6,12,24 h적주기성장응력.매분종10개순배,매순배포괄3 s견장,3 s송이.Hoechst 33258염색관찰세포적형태학변화;류식세포의검측세포조망정황;RT-PCR법검측촉조망기인bax mRNA적표체;Western blot검측신호통로중p38MAPK화p-p38MAPK단백적표체.결과여결론:수착가력시간적연장,세포축점출현핵고축급조망소체,조망솔증가(P < 0.05),bax mRNA표체증다(P < 0.05);세포p38MAPK화p-p38MAPK단백균재가력6 h체도최저,차후축점승고.p38MAPK억제제SB203580가억제가력인기적세포조망,감소bax mRNA급p38MAPK화p-p38MAPK단백적표체(P < 0.05).설명p38MAPK신호통로재응력개도적성기세포조망중기도중요적작용.
BACKGROUND: Because of complicated physiological environment and difficulty to control experimental conditions, it is difficult to get satisfactory results from in vivo studies of cell mechanics.OBJECTIVE: To study the action and mechanism of p38MAPK signaling pathways on myoblast apoptosis based on successful construction of in vitro mechanical stimulation models.METHODS: The C2C12 cells cultured in vitro were divided into control group and SB203580 treatment group. Cyclic tensile stress was applied on the C2C12 myoblast cells for 0, 6, 12 and 24 hours in each group. The Flexcell Strain Unit-5000T was used to expose C2C12 myoblast cell to an equiaxial cyclic of 15% magnitude and a frequency of 10 cycles/min, each cycle including the 3 s stretch and 3 s relaxation. Hoechst 33258 fluorescent staining and optical microscope were used to detect cell apoptosis. RT-PCR, flow cytometric analysis were used to observe the apoptosis of C2C12 myoblast cells and Western blotting were used to detect the activity of p38MAPK and p-p38MAPK. RESULTS AND CONCLUSION: The optical microscope tested the change in the morphology. Hoechst 33258 staining showed that after treatment with cyclic stress, the cell took the typical appearance of apoptosis with chromatin condensation and apoptotic bodies. RT-PCR and flow cytometry showed that with the extension of time the rate of the apoptosis of C2C12 myoblast cell increased. And cells imposed SB203580 before imposing cyclical tensile stress, the results showed that the apoptosis was markedly affected, and the p-p38MAPK expression declined apparently. These findings demonstrate that p38MAPK signaling pathways in stress mediated into C2C12 myoblast cell apoptosis plays an important role.