CAJ | 학술논문

目的观察小干扰RNA技术(siRNA)抑制核纤层蛋白A/C(lamin A/C)表达对牵张刺激下小鼠前成骨细胞MC3T3-E1增殖及凋亡的影响.方法化学合成3条针对lamin A/C靶点的siRNA序列,脂质体转染MC3T3-E1细胞,实时定量聚合酶链反应(PCR)和Western blot检测lamin A/C mRNA和蛋白变化.细胞转染72 h后行牵张刺激6 h,Hoechst 33258检测DNA含量来反映细胞增殖,流式细胞术检测细胞周期及凋亡.结果筛选出的siRNA-2显著抑制lamin A/C mRNA和蛋白产物表达(P＜0.01).未转染细胞经牵张刺激,DNA合成随时间推移而增加;细胞G0/G1期比例为65.19%;S期为22.57%,细胞凋亡率为11.49%;转染细胞经牵张刺激后,DNA合成较未转染细胞显著性减少(P＜0.01);G0/G1期比例提高至85.82%;S期降低至11.37%,凋亡率达19.32%(P＜0.01).结论抑制lamin A/C表达会导致牵张刺激诱导的促增殖作用减弱,细胞周期阻滞于G0/G1期,促进细胞的凋亡.
목적 관찰소간우RNA기술(siRNA)억제핵섬층단백A/C(lamin A/C)표체대견장자격하소서전성골세포MC3T3-E1증식급조망적영향.방법 화학합성3조침대lamin A/C파점적siRNA서렬,지질체전염MC3T3-E1세포,실시정량취합매련반응(PCR)화Western blot검측lamin A/C mRNA화단백변화.세포전염72 h후행견장자격6 h,Hoechst 33258검측DNA함량래반영세포증식,류식세포술검측세포주기급조망.결과 사선출적siRNA-2현저억제lamin A/C mRNA화단백산물표체(P＜0.01).미전염세포경견장자격,DNA합성수시간추이이증가;세포G0/G1기비례위65.19%;S기위22.57%,세포조망솔위11.49%;전염세포경견장자격후,DNA합성교미전염세포현저성감소(P＜0.01);G0/G1기비례제고지85.82%;S기강저지11.37%,조망솔체19.32%(P＜0.01).결론 억제lamin A/C표체회도치견장자격유도적촉증식작용감약,세포주기조체우G0/G1기,촉진세포적조망.
Objective To investigate the effect of lamin A/C inhibition using siRNA technology on proliferation and apoptosis of MC3T3-E1 cells exposed to the stretch stimulus. Methods Three chemically synthesized siRNAs targeting lamin A/C were transfected into MC3T3-E1 cell by lipofectamineTM 2000. The most efficient siRNA inhibiting lamin A/C mRNA and protein expression was identified by realtime polymerase chain reaction (PCR) and Western blotting respectively. After 72 h siRNA transfection,MC3T3-E1 cells were subsequently subjected to 2500 με stretch for 6 h with four-point-bending system.DNA content was detected by Hoechst 33258 fluorochrome to evaluate cell proliferation. Flow cytometry was employed to investigate the changes in cell cycle and apoptosis of MC3T3-E1 cells. Results The siRNA-2 was identified to inhibit lamin A/C mRNA and protein expression significantly (P ＜ 0. 01 ) and utilized in the following experiment. DNA assay showed that stretch stress led to an increased DNA synthesis in a time-dependent manner in non-transfected cells, while the similar increase in DNA content was not displayed in siRNA transfected cells applied with the same mechanical stimulus (P ＜ 0. 01 ). Compared with non-transfected MC3T3-E1 cells, stretch stress increased the percentage of G0/G1 phase in transfected cells from 65. 19% to 85.82%, and decreased the percentage of S phase from 22. 57% to 11.37%. At the meantime, apoptosis rate was significantly increased from 11.49% to 19. 32% ( P ＜ 0. 01 ). Conclusion Inhibition of lamin A/C expression attenuates the mitogenic effects of MC3T3-E1 exposed to stretch stress,blocks more cells arrest in G0/G1 phase and increases cell apoptosis.