CAJ | 학술논문

目的：研究自噬在As2 O3诱导的Raji细胞死亡中的作用及机制。方法透射电镜和MDC荧光染色观察细胞自噬形态,MTT 法测定细胞增殖活性；流式细胞术( FCM )及FITC-Annexin-V/PI染色检测细胞周期和细胞凋亡, Western blot 检测细胞LC3-Ⅰ/Ⅱ表达及转换；RT-PCR检测细胞bcl-2 mRNA和p53 mRNA的表达水平。结果 As2 O3明显抑制Raji细胞增殖,诱导其细胞周期G2/M阻滞和凋亡,抑制bcl-2基因和增强p53基因表达。同时, As2 O3可同步诱发Raji细胞的自噬活性。自噬抑制剂3-甲基腺嘌呤(3-MA)抑制As2 O3诱导的自噬活性,上调bcl-2基因和下调p53基因表达,抑制As2 O3对 Raji 细胞的杀伤效应、降低 Raji 细胞对As2 O3的敏感性。而自噬诱导剂雷帕霉素( Rapa)的作用则与3-MA的效应正好相反。结论细胞凋亡和自噬性细胞死亡共存于As2 O3诱导的Raji淋巴瘤细胞死亡中,Bcl-2和p53可能起着关键的调控作用。
목적：연구자서재As2 O3유도적Raji세포사망중적작용급궤제。방법투사전경화MDC형광염색관찰세포자서형태,MTT 법측정세포증식활성；류식세포술( FCM )급FITC-Annexin-V/PI염색검측세포주기화세포조망, Western blot 검측세포LC3-Ⅰ/Ⅱ표체급전환；RT-PCR검측세포bcl-2 mRNA화p53 mRNA적표체수평。결과 As2 O3명현억제Raji세포증식,유도기세포주기G2/M조체화조망,억제bcl-2기인화증강p53기인표체。동시, As2 O3가동보유발Raji세포적자서활성。자서억제제3-갑기선표령(3-MA)억제As2 O3유도적자서활성,상조bcl-2기인화하조p53기인표체,억제As2 O3대 Raji 세포적살상효응、강저 Raji 세포대As2 O3적민감성。이자서유도제뢰파매소( Rapa)적작용칙여3-MA적효응정호상반。결론세포조망화자서성세포사망공존우As2 O3유도적Raji림파류세포사망중,Bcl-2화p53가능기착관건적조공작용。
Aim To investigate the role of autophagy and its mechanism in Raji cell death induced by arse-nic trioxide. Methods Transmission electron micros-copy ( SEM) and MDC fluorescence staining were used to observe autophagy. MTT colorimetry was employed to assay the cellular proliferating activity. Cell apopto-sis and cell cycle analysis were performed using FITC-Annexin-V/PI double staining and flow cytometry ( FCM) . The expressions of LC3 and the conversion of LC3-I to LC3-II were measured by western bloting. The expression of bcl-2 mRNA and p53 mRNA were detected by reverse transcription-polymerase chain re-action ( RT-PCR ) . Results Arsenic trioxide could obviously inhibit the proliferation of Raji cells, arrest the cells at G2/M phase and induce apoptosis. Mean-while, arsenic trioxide markedly inhibited the expres-sion of bcl-2 mRNA and enhanced the expression of p53 mRNA in Raji cells. Arsenic trioxide also induced autophagy synchronously which paralleled with the in-duction of apoptosis in Raji cells, and 3-MA, an auto-phagy inhibitor, was able to reverse the arsenic triox-ide-activated autophagic activity, up-regulate bcl-2, down-regulated p53 expression and suppress the lethal effect of arsenic trioxide on Raji cells to reduce their sensitivity to arsenic trioxide. In contrast, the Rapamy-cin, an autophagy inducer, possessed the completely opposite effects on Raji cells compared with 3-MA. Conclusions The apoptosis and autophagic cell death are coexistent in arsenic trioxide-triggered death of Raji lymphoma cells, and Bcl-2 and p53 may play a key regulating role in this process.