CAJ | 학술논문

目的研究蛋白酶体抑制剂硼替佐米(bortezomi)对白血病细胞株K562细胞的增殖、凋亡及X连锁凋亡抑制蛋白(XIAP)表达的影响.方法将不同浓度的硼替佐米作用于K562细胞,采用WST-1法测定细胞增殖活性,瑞特-吉姆萨染色观察细胞形态学变化,用流式细胞术、原位末端转移酶标记(TUNEL)法检测细胞凋亡,RT-PCR法检测XIAP mRNA表达,SP免疫组化法检测XIAP蛋白的表达.结果 5、10、30、50、100 nmol/L硼替佐米作用于K562细胞24 h,细胞增殖抑制率分别为(13.6±0.2)%、(28.7±0.5)%、(55.4±1.1)%、(68.1±1.1)%、(81.4±0.1)%,空白对照组为(1.2±0.0)%(P＜0.05),24 h IC50值为24.6 nmol/L.30 nmoL/L硼替佐米作用于K562细胞12、24、36、48 h,细胞增殖抑制率分别为(29.1±0.9)%、(55.4±1.1)%、(57.8±0.8)%、(59.8±1.2)%,12 h组与24 h组比较,差异有统计学意义(P＜0.05),但24 h组与36、48 h组比较差异有统计学意义(P＞0.05);30 nmol/L硼替佐米作用于K562细胞24 h,镜下可见细胞核固缩、核边集、核碎裂,胞质中见大量空泡及凋亡小体,对照组细胞形态正常.TUNEL检测法显示凋亡细胞阳性率为49.0%,空白对照组阳性率2.3%(P＜0.05).10、30、100 nmoL/L硼替佐米作用于K562细胞24 h,AnnexinⅤ-FTTC/PI双标法显示其凋亡率分别为(32.2±1.2)%、(53.9±1.3)%和(81.3±2.8)%,均高于对照组(0.3±0.6)%(P＜0.05).RT-PCR法检测结果显示随硼替佐米浓度增高,XIAP mRNA表达下调明显.免疫组化结果显示硼替佐米组XIAP蛋白表达下调,与空白对照组比较差异有统计学意义(P＜0.05).结论硼替佐米呈时间、浓度依赖性抑制K562细胞增殖,并可能通过下调XIAP的表达诱导细胞凋亡.
목적 연구단백매체억제제붕체좌미(bortezomi)대백혈병세포주K562세포적증식、조망급X련쇄조망억제단백(XIAP)표체적영향.방법 장불동농도적붕체좌미작용우K562세포,채용WST-1법측정세포증식활성,서특-길모살염색관찰세포형태학변화,용류식세포술、원위말단전이매표기(TUNEL)법검측세포조망,RT-PCR법검측XIAP mRNA표체,SP면역조화법검측XIAP단백적표체.결과 5、10、30、50、100 nmol/L붕체좌미작용우K562세포24 h,세포증식억제솔분별위(13.6±0.2)%、(28.7±0.5)%、(55.4±1.1)%、(68.1±1.1)%、(81.4±0.1)%,공백대조조위(1.2±0.0)%(P＜0.05),24 h IC50치위24.6 nmol/L.30 nmoL/L붕체좌미작용우K562세포12、24、36、48 h,세포증식억제솔분별위(29.1±0.9)%、(55.4±1.1)%、(57.8±0.8)%、(59.8±1.2)%,12 h조여24 h조비교,차이유통계학의의(P＜0.05),단24 h조여36、48 h조비교차이유통계학의의(P＞0.05);30 nmol/L붕체좌미작용우K562세포24 h,경하가견세포핵고축、핵변집、핵쇄렬,포질중견대량공포급조망소체,대조조세포형태정상.TUNEL검측법현시조망세포양성솔위49.0%,공백대조조양성솔2.3%(P＜0.05).10、30、100 nmoL/L붕체좌미작용우K562세포24 h,AnnexinⅤ-FTTC/PI쌍표법현시기조망솔분별위(32.2±1.2)%、(53.9±1.3)%화(81.3±2.8)%,균고우대조조(0.3±0.6)%(P＜0.05).RT-PCR법검측결과현시수붕체좌미농도증고,XIAP mRNA표체하조명현.면역조화결과현시붕체좌미조XIAP단백표체하조,여공백대조조비교차이유통계학의의(P＜0.05).결론 붕체좌미정시간、농도의뢰성억제K562세포증식,병가능통과하조XIAP적표체유도세포조망.
Objective To investigate the effect of proteasome inhibitor bortezomib on proliferation,apoptosis of K562 cells and the expression of XIAP. Methods K562 cells were treated with bortezomib at different concentration. Cell proliferation was analyzed by WST-1 assay, cell apoptosis by flow cytometry and TUNEL, XIAP mRNA expression from 5 - 100 nmol/L by RT-PCR, and XIAP protein expression by SP immunohistochemistry. Results K562 cells were treated with bortezomib at different concentrations for 24 h respectively, the cells growth was significantly inhibited with inhibition rates from( 13.6 ±0.2)% to (81.4 ±0. 1 ) %, respectively, being markedly higher than that of control ( 1.2 ± 0. 1 ) % ( P ＜ 0. 05 ). IC50 was 24.6 nmol/L of bortezomib treated for 24 h. When K562 cells were treated with 30 nmol/L of bortezomib for 12 - 48 h, the inhibition rates were (29.1 ± 0. 9) % to (59.8 ± 1.2 ) %, respectively, the differences being statistically significant( P ＜0.05 ) between 12 h group and 24 h group, while there was no statistical difference between 24 h, 36 h and 48 h groups. K562 cells treated with 30 nmol/L bortezomib for 24 h showed nuclear condensation, nuclear margination, nuclear fragmentation, cytoplasmic vacuoles and a large number of apoptotic body formation. The apoptotic cells rate was 83.67% in bortezomib treated group, and 2.33% in untreated group (P ＜ 0.05 ). The expression of XIAP mRNA was decreased in a dose-dependent manner, and the expression of its protein was down-regulated. Conclusion Bortezomib can inhibit the proliferation of K562 cells, and induce apoptosis by down-regulating the expression of XIAP, providing the laboratory evidence for the targeted therapy in acute leukemia.