CAJ | 학술논문

目的：研究曲古抑菌素（TSA）对HepG2细胞凋亡的影响及可能的机制。方法取对数生长期HepG2细胞，TSA（50~500 nmol/L）诱导，同时设空白对照组，用CCK-8检测细胞增殖影响；流式细胞术检测细胞周期；Annexin V-FTIC/PI双染细胞检测凋亡；倒置显微镜观察细胞形态并采图；Western blot检测HepG2细胞中β-catenin、HDAC1、HDAC3、H3K9、CyclinD1、Bax等蛋白的表达。定量PCR方法检测HDAC1，HDAC3基因的表达。结果 CCK-8检测结果显示TSA对肝癌HepG2细胞生长抑制呈剂量和时间依赖性；流式细胞术检测细胞周期表明，药物组细胞G0/G1期所占比例增加，S期所占比例下降，G2/M所占比例增加；细胞凋亡检测表明，空白对照组凋亡率（6.22±0.25）%；250 nmol/L组凋亡率（7.17±0.20）%；500 nmol/L组凋亡率（18.14±0.42）%；倒置显微镜下观察可见，随着时间推移空白对照组细胞生长良好；而药物组细胞则出现变形，有大量漂浮细胞；Western blot检测结果显示β-catenin、H3K9和Bax蛋白表达水平均明显升高，CyclinD1、HDAC1、HDAC3的表达水平则呈下降趋势；定量PCR结果显示：HDAC1、HDAC3基因的表达无明显变化。结论 TSA可通过抑制HDAC的活性，促进组蛋白乙酰化，活化Wnt/β-catenin信号通路来发挥其抑制HepG2细胞增殖，诱导周期阻滞及凋亡的作用。
목적：연구곡고억균소（TSA）대HepG2세포조망적영향급가능적궤제。방법취대수생장기HepG2세포，TSA（50~500 nmol/L）유도，동시설공백대조조，용CCK-8검측세포증식영향；류식세포술검측세포주기；Annexin V-FTIC/PI쌍염세포검측조망；도치현미경관찰세포형태병채도；Western blot검측HepG2세포중β-catenin、HDAC1、HDAC3、H3K9、CyclinD1、Bax등단백적표체。정량PCR방법검측HDAC1，HDAC3기인적표체。결과 CCK-8검측결과현시TSA대간암HepG2세포생장억제정제량화시간의뢰성；류식세포술검측세포주기표명，약물조세포G0/G1기소점비례증가，S기소점비례하강，G2/M소점비례증가；세포조망검측표명，공백대조조조망솔（6.22±0.25）%；250 nmol/L조조망솔（7.17±0.20）%；500 nmol/L조조망솔（18.14±0.42）%；도치현미경하관찰가견，수착시간추이공백대조조세포생장량호；이약물조세포칙출현변형，유대량표부세포；Western blot검측결과현시β-catenin、H3K9화Bax단백표체수평균명현승고，CyclinD1、HDAC1、HDAC3적표체수평칙정하강추세；정량PCR결과현시：HDAC1、HDAC3기인적표체무명현변화。결론 TSA가통과억제HDAC적활성，촉진조단백을선화，활화Wnt/β-catenin신호통로래발휘기억제HepG2세포증식，유도주기조체급조망적작용。
Objective To investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism. Methods HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR. Results Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)%and (18.14 ± 0.42)%after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes. Conclusion TSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.