CAJ | 학술논문

目的：研究DADS诱导人白血病K562细胞凋亡的分子机制。方法应用MTT法检测细胞的活性；流式细胞术检测细胞内的活性氧( reactive oxygen species, ROS)水平以及凋亡细胞百分率；Real-time PCR 检测NADPH氧化酶各亚基mRNA水平；免疫共沉淀检测蛋白 Rac2与蛋白 p67phox的结合；Westernblot检测Rac2蛋白的表达。结果 DADS能明显抑制K562细胞的增殖,呈时间和剂量依赖性；6 mg ·L-1 DADS 作用人白血病K562细胞6 h后,NADPH氧化酶复合物的6个亚基mRNA水平都明显上调；5.0、10.0 mg ·L-1 DADS作用人白血病K562细胞24 h后蛋白 Rac2的表达水平明显上调；免疫共沉淀结果显示, DADS 诱导的K562细胞凋亡过程中有Rac2与p67phox结合；流式细胞术检测凋亡细胞百分率结果显示, PMA能明显提高DADS诱导K562细胞凋亡的作用,而DPI能抑制DADS诱导K562细胞凋亡。 PMA能提高DADS诱导K562细胞活性氧的水平,而DPI明显抑制了活性氧的产生。结论 NADPH氧化酶的活化是DADS诱导K562细胞凋亡过程中活性氧的主要来源,DADS通过活化NADPH氧化酶诱导K562细胞凋亡。
목적：연구DADS유도인백혈병K562세포조망적분자궤제。방법응용MTT법검측세포적활성；류식세포술검측세포내적활성양( reactive oxygen species, ROS)수평이급조망세포백분솔；Real-time PCR 검측NADPH양화매각아기mRNA수평；면역공침정검측단백 Rac2여단백 p67phox적결합；Westernblot검측Rac2단백적표체。결과 DADS능명현억제K562세포적증식,정시간화제량의뢰성；6 mg ·L-1 DADS 작용인백혈병K562세포6 h후,NADPH양화매복합물적6개아기mRNA수평도명현상조；5.0、10.0 mg ·L-1 DADS작용인백혈병K562세포24 h후단백 Rac2적표체수평명현상조；면역공침정결과현시, DADS 유도적K562세포조망과정중유Rac2여p67phox결합；류식세포술검측조망세포백분솔결과현시, PMA능명현제고DADS유도K562세포조망적작용,이DPI능억제DADS유도K562세포조망。 PMA능제고DADS유도K562세포활성양적수평,이DPI명현억제료활성양적산생。결론 NADPH양화매적활화시DADS유도K562세포조망과정중활성양적주요래원,DADS통과활화NADPH양화매유도K562세포조망。
Aim Toresearchthemolecularmecha-nisms of DADS-induced apoptosis in human leukemia K562cells.Methods Cellviabilitywasmeasuredby MTT. Levels of DADS-induced ROS were measured by 2ˊ, 7ˊ-dichlorofluorescein diacetate ( DCFH-DA) fluo-rescence. DADS-induced mRNA levels of components of the NADPH oxidase were detected by Real-time PCR. The combination of protein Rac2 and p67phox was measured by immunoprecipitation assays. Flow cy-tometry methods were used to determine the percentage of apoptosis cells. DADS-induced Rac2 levels were measuredbyWesternblot.Results TheDADS-trea-ted K562 cells showed a dose-and time-dependent de-crease in cell viability and proliferation. There was sig-nificant up-regulation of the mRNA level of components of the NADPH oxidase complex in K562 cells after treatment with 6 mg·L-1 DADS for 6 h. Western blot results revealed that, compared with the control group, there was a significant up-regulation of Rac2 protein in K562 cells treated with 5. 0 and 10. 0 mg·L-1 DADS for 24h. And Rac2 combined with p67phox in DADS-induced apoptosis in K562 cells. PMA markedly in-creased the percentage of apoptotic cells, and DPI re-duced the percentage of apoptotic cells in DADS-in-duced K562 cells. Levels of DADS-induced ROS, also showed enhancement when exposed in PMA, but there was no DADS-induced ROS production evident when exposed in DPI in DADS induced K562 cells. Conclu-sions TheseresultsindicatethatNADPHoxidaseis the main source of DADS-induced ROS production. Diallyl disulfide induces apoptosis in human leukemia K562 cells through activation of NADPH oxidase.