CAJ | 학술논문

目的:探讨抗人死亡受体5(DRS)功能性单克隆抗体(mDRA-6)对Jurkat细胞诱导凋亡的线粒体途径的分子机制.方法:MTT法检测单克隆抗体(mDRA-6)对Jurkat细胞生长抑制作用的剂量-效果及时间-效果关系;JC-1单染流式细胞术定量分析技术对凋亡的Jurkat细胞线粒体膜电位的变化进行分析;免疫印迹技术检测凋亡细胞的Caspase-8、3、9及Bid、Bax、Bcl-2、Cyto c等凋亡相关蛋白的表达情况.结果:mDRA-6对Jurkat细胞抑制具有明显剂量-效果和时间-效果关系;流式细胞仪检测显示,2.0μg,/ml浓度的mDRA-6作用15、30、60和120分钟时,Jurkat细胞线粒体膜电位改变率分别为20.14%、19.34%、21.11%、30.90%,呈逐渐增高趋势.免疫印迹技术检测显示,mDRA-6作用后,Jurkat细胞Caspase-8、9及Bid、Bax、Bcl-2、Cyto c等表达活性增加,并且Cyto C在线粒体内为递减而在胞浆呈递增的表达现象.结论:mDRA-6通过激活外源性膜受体,继而启动细胞线粒体途径导致Jurkat细胞凋亡.本研究为mDRA-6对白血病治疗奠定实验基础.
목적:탐토항인사망수체5(DRS)공능성단극륭항체(mDRA-6)대Jurkat세포유도조망적선립체도경적분자궤제.방법:MTT법검측단극륭항체(mDRA-6)대Jurkat세포생장억제작용적제량-효과급시간-효과관계;JC-1단염류식세포술정량분석기술대조망적Jurkat세포선립체막전위적변화진행분석;면역인적기술검측조망세포적Caspase-8、3、9급Bid、Bax、Bcl-2、Cyto c등조망상관단백적표체정황.결과:mDRA-6대Jurkat세포억제구유명현제량-효과화시간-효과관계;류식세포의검측현시,2.0μg,/ml농도적mDRA-6작용15、30、60화120분종시,Jurkat세포선립체막전위개변솔분별위20.14%、19.34%、21.11%、30.90%,정축점증고추세.면역인적기술검측현시,mDRA-6작용후,Jurkat세포Caspase-8、9급Bid、Bax、Bcl-2、Cyto c등표체활성증가,병차Cyto C재선립체내위체감이재포장정체증적표체현상.결론:mDRA-6통과격활외원성막수체,계이계동세포선립체도경도치Jurkat세포조망.본연구위mDRA-6대백혈병치료전정실험기출.
Objective:To investigate mitochondrial-dependent molecular mechanisms of a novel agonistic anti-human death receptor 5 (DR5) monoclonal antibody(mDRA-6) inducing apoptosis in Jurkat cell.Methods:The dose-dependent and time-dependent cell growth suppression of mDRA-6 in Jurkat cells was determined by MTT assay.The measurement of the mitochondrial transmembrane potential(ΔΨm) of Jurkat cells was detected by flow cytometry with JC-1 single staining.Caspase-8,9 as well as Bid,Bax,Bcl-2 and Cyto c of apoptotic Jurkat cells were analyzed by Western blot after mDRA-6 treatment.Results:The mDRA-6 induced cell growth suppression and cytotoxicity in dose-dependent manner and time-dependent manner.After mDRA-6 treatment at 2.0 μg/ml for15 min,30 min,60 min and 120 min,the change in ΔΨm were 20.14 %,19.34 %,21.11% and 30.90% respectively by JC-1 single staining.Western blot revealed that the level of active fragments of Caspase-8,9 and Bid,Bax,Bcl-2 and Cyto c respectively,and the amount of Cyto c was increased in cytosol concomitant with the related attenuation of Cyto c in mitochondria.Conclusion: Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR5 ligatian to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated,and endogenic mitochondrial-dependent cell apoptosis pathway is activated.mDRA-6 may be a useful agent in investigating human leukemia therapy by using TRAIL/DR5.