CAJ | 학술논문

目的研究阿霉素诱导白血病细胞凋亡的剂量及时间关系,探索其相关的分子机制.方法分别以0 1、0.2、0.5、1.0 mg/L的阿霉素处理人类Jurkat白血病细胞6、12、24、48 h.其中一份样本在加入0 2 mg/L阿霉素前用zVAD-fmk(苄氧羰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮)预处理.应用AnV/PI双染细胞,在流式细胞仪上分析AnV/PI双阳性的凋亡细胞.采用WesternBlot技术检测FasL和FADD(Fas相关死亡域)的表达.结果6h时所有剂量的阿霉素诱导的凋亡细胞无显著差异(P＞0.05),在12 h,只有1.0 mg/L诱导细胞明显凋亡.当细胞与0 2和0 5 mg/L的阿霉素共同培养24 h或36 h,观察到调亡细胞显著增加(P＜0 001).在zVAD-fmk存在的情况下,当细胞与阿霉素一同培养时,由阿霉素诱导的细胞凋亡完全受到抑制(P＜0.001).随着阿霉素作用时间增加,FasL和FADD表达水平相应增加.结论在阿霉素诱导的白血病细胞凋亡中,阿霉素以剂量和时间依赖方式诱导细胞凋亡;上调FasL可能启动FasL信号通路的激话,而caspase是最终的执行者.
목적연구아매소유도백혈병세포조망적제량급시간관계,탐색기상관적분자궤제.방법분별이0 1、0.2、0.5、1.0 mg/L적아매소처리인류Jurkat백혈병세포6、12、24、48 h.기중일빈양본재가입0 2 mg/L아매소전용zVAD-fmk(변양탄-힐안선-병안선-천동안선-불갑기동)예처리.응용AnV/PI쌍염세포,재류식세포의상분석AnV/PI쌍양성적조망세포.채용WesternBlot기술검측FasL화FADD(Fas상관사망역)적표체.결과6h시소유제량적아매소유도적조망세포무현저차이(P＞0.05),재12 h,지유1.0 mg/L유도세포명현조망.당세포여0 2화0 5 mg/L적아매소공동배양24 h혹36 h,관찰도조망세포현저증가(P＜0 001).재zVAD-fmk존재적정황하,당세포여아매소일동배양시,유아매소유도적세포조망완전수도억제(P＜0.001).수착아매소작용시간증가,FasL화FADD표체수평상응증가.결론재아매소유도적백혈병세포조망중,아매소이제량화시간의뢰방식유도세포조망;상조FasL가능계동FasL신호통로적격화,이caspase시최종적집행자.
Objective To investigate the dose and time kinetics of induction of apoptosis induced by doxorubicin in J urkat leukemiacells, and to explore its pertinent molecular mechanisms. Methods Human Jurkat leukemia T - cells were treated with doxorubicin at theconcentration of 0.1 mg/L, 0.2 mg/L, 0.5 mg/L and 1.0 mg/L for 6,12,24 and 36 hours, respectively, of which one sample was pre-treated with zVAD- fmk (benzyloxycarbonyl - Val -Ala - Asp - fluoromethylketone) prior to addition of doxorubicin 0.2 mg/L. Apop-tosis was detected with both annexin V - FITC and propidium iodide ( PI ) staining and annexin V FITC and PI double positive cellswere analyzed by flow cytometry. Western blot was used to evaluate the level of Fas ligand (FasL) and FADD (Fas - associated death do-main) expression. Results The differences of apoptotic cells induced by all dose of doxorubicin were not significant (P＞0.05 ) at 6hour;at 12 hour, only the highest dose, 1 mg/L, significantly induced cell apoptosis;while the lowest dose,0.1 mg/L, did not significantlycaused cell apoptosis for all time points. After exposure to the doses of 0.2 and 0.5 mg/L for 24 or 36 hours,a significant increase in per-centage of apoptotic cells was observed (P ＜ 0.001 ). Apoptosis induced by doxorubicin was completely inhibited when the cells were incu-bated with doxorubicin in the presence of zVAD - fmk (P ＜ 0. 001 ). The level of FasL and FADD expression correspondingly increasedwith exposure time to doxorubicin. Conclusions Doxorubicin induces apoptosis in a dose - and time - dependent manner; upregulatedFasL may initiate the activation of the Fas signaling pathway and caspases are the ultimate executioner in the induction of leukemia cellapoptosis by doxorubicin.