scFvCD20:sTRAIL蛋白诱导BJAB细胞凋亡的实验研究  

Study on apoptosis of BJAB cells induced by fusion protein scFvCD20:sTRAIL

作　　者：杨雨琪[1] 张晓龙[2] 张砚君[2] 吕军强[1] 孙睿声 安鹏姣 于楠楠[1] 

机构地区：[1]天津医科大学药学院基础医学院,天津300070 [2]中国医学科学院北京协和医学院血液病医院血液研究所实验血液学国家重点实验室,天津300020

出　　处：《现代免疫学》2016年第3期183-188,共6页Current Immunology

基　　金：国家自然科学基金资助项目(81400176);天津市应用基础与前沿技术研究计划资助项目(14JCYBJC23700)

摘　　要：研究融合蛋白scFvCD20:sTRAIL诱导B淋巴瘤细胞BJAB凋亡的发生以及凋亡相关蛋白的表达变化。采用PCR、重叠PCR、酶切、连接等方法构建pLentiR.scFvCD20:sTRAIL、pLentiR.ISZ-sTRAIL、pLentiR.scFvCD20及pLentiR.CopGFP慢病毒表达载体,瞬时转染293T细胞获得含有scFvCD20:sTRAIL融合蛋白的细胞培养上清,采用CCK8细胞增殖抑制实验检测scFvCD20:sTRAIL融合蛋白对CD20阳性BJAB细胞的生长抑制作用,流式细胞仪检测其对BJAB细胞诱导凋亡,Western blotting检测凋亡过程中内源性及外源性凋亡信号传导途径相关蛋白的变化。结果显示,成功构建了慢病毒表达载体pLentiR.scFvCD20:sTRAIL、pLentiR.ISZ-sTRAIL、pLentiR.scFvCD20及pLentiR.CopGFP,瞬时转染293T细胞后并可获得融合蛋白的表达。与ISZ-sTRAIL比较,融合蛋白scFvCD20:sTRAIL可不同程度地提高对CD20阳性BJAB细胞的生长抑制作用,凋亡细胞增多,其中Bcl-2表达下降、Bax表达升高,Caspase 8、Caspase 9、Caspase 3及PARP被特异性剪切活化。研究表明,融合蛋白scFvCD20:sTRAIL可诱导BJAB细胞凋亡,与bcl-2/bax的表达变化以及Caspase的剪切活化有关。This study aimed to the investigation of the apoptosis of B-cell lymphoma cell line BJAB induced by our fusion pro- tein scFvCD20：sTRAIL. The methods of PCR, overlap PCR, restriction enzyme cleavage and linkage were used to construct lentivirus expression vectors including pLentiR, scFvCD20：sTRAIL, pLentiR. ISZ： sTRAIL, pLentiR, scFvCD20 and pLentiR. CopGFP. Fusion protein scFvCD20：sTRAIL was collected from the supernatant of 293T cells transiently transfected with the corresponding vector. CCK8 assay was applied to detect the antigen-restricted cell death induced by scFvCD20： sTRAIL in CD20-positive BJAB cells. The apoptosis of BJAB cells was inspected by flow cytometry analysis and changes of proteins in- volved in both extrinsic and intrinsic apoptotic pathway were identified by Western blotting. We successfully constructed the lentivirus expression vectors pLentiR, scFvCD20 ： sTRAIL, pLentiR. ISZ ： sTRAIL, pLentiR, scFvCD20 and pLentiR. CopGFP. Exposure to scFvCD20 ：sTRAIL fusion protein produced a potent inhibition of proliferation in CD20-positive BJAB cells. Apop- tosis was markedly augmented through modulation of both extrinsic and intrinsic apoptosis signalings. The levels of Bcl-2 pro rein decreased and Bax increased whereas Caspase-8, 9, 3 and PARP was cleaved actively. The results indicated that fusion protein scFvCD20：sTRAIL could induce apoptosis of BJAB cells, which was associated with changes of expression of Bcl-2, Bax as well as activation of Caspases and PARP.

关 键 词：CD20 TRAIL B淋巴细胞 凋亡 蛋白表达 

分 类 号：R392.5[医药卫生—免疫学]