26S蛋白酶抑制剂b-AP15对Jurkat及RS4;11细胞增殖、凋亡的影响及凋亡机制的研究  

Role of the 26S proteasome inhibitor b-AP15 on Jurkat cells and RS4;11 cells

作　　者：周俊[1] 曹江[1] 孟凡静[1] 冯浩[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,221002

出　　处：《国际输血及血液学杂志》2015年第3期185-191,共7页International Journal of Blood Transfusion and Hematology

基　　金：国家自然科学基金资助项目（81100349）;江苏省“六大人才高峰”项目（2011-WS-067）

摘　　要：目的 探讨26S蛋白酶抑制剂b-AP15对Jurkat细胞与RS4;11细胞增殖及细胞凋亡的影响,并分析其作用机制.方法 无菌条件下对Jurkat细胞与RS4;11细胞进行培养,根据有无b-AP15处理,将其分为4组：①Jurkat细胞实验组;②Jurkat细胞对照组;③RS4;11细胞实验组;④RS4;11细胞对照组.其中,Jurkat细胞实验组与RS4;11细胞实验组采用浓度分别为0.05,0.10,0.50,1.00,5.00 μmol/L b-AP15处理24,48 h;Jurkat细胞对照组与RS4;11细胞对照组采用RPMI 1640培养基进行同步培养.采用CCK-8法绘制4组细胞增殖曲线;采用Annexin Ⅴ-别藻蓝蛋白（APC）/7-氨基放线菌素D（7-AAD）双标记流式细胞术检查4组细胞凋亡情况;采用流式细胞术检测细胞4组细胞周期分布情况;采用实时定量PCR检测4组细胞B细胞淋巴瘤/白血病-2相关Ⅹ蛋白（Bax）基因、B细胞淋巴瘤/白血病（Bcl）-2基因、Ⅹ连锁凋亡抑制蛋白（XIAP）、周期素依赖性激酶（CDK）1基因及半胱氨酸天冬氨酸蛋白酶（caspase）-3基因mRNA表达水平.结果 ①b-AP15对Jurkat细胞与RS4;11细胞增殖均具有抑制作用,且该抑制效应呈时间与剂量依赖性;Jurkat细胞经b-AP15处理24,48 h后,半数抑制浓度（IC50）分别为（1.52±0.35）μmol/L与（0.54±0.01） μmol/L,RS4;11细胞经b-AP15处理24,48 h后IC50分别为（0.97±0.02）μmol/L与（0.08±0.03）μmol/L.②使用浓度为1.5 μmol/L b-AP15处理Jurkat细胞实验组后,其与Jurkat细胞对照组相比细胞凋亡率显著增高,且差异有统计学意义（P＜0.001）;使用浓度为1.0 μmol/Lb AP15处理RS4;11细胞实验组后,其与RS4;11细胞对照组相比细胞凋亡率亦显著增加,且差异亦有统计学意义（P＜0.001）;且Jurkat细胞与RS4;11细胞经b-AP15处理48 h后,与处理24 h后者相比细胞凋亡率增高,且差异均有统计学意义（t=11.887,7.449;P＜0.001）.③与Jurkat细胞对照组相比,使用浓度为1.5 μmol/L b-AP15处理Jurkat细胞实验组24 h后,Jurkat细胞周Objective To investigate the effect of 26S proteasome inhibitor b-AP15 on proliferation and apoptosis of Jurkat cells and RS4;11 cells and to explore its possible mechanism.Methods J urkat cells and RS4;11 cells were cultured under asepsis condition.According to the presence of b-AP15 during culturing,Jurkat cells and RS4;11 cells were divided into four groups：①Jurkat cells experimental group,②Jurkat cells control group,③RS4;11 cells experimental group,④RS4;11 cells control group.Jurkat cells experimental group and RS4;11 cells experimental group were cultured with 0.05,0.10,0.50,1.00,5.00 μmol/L b-AP15 for 24 h and 48 h,while Jurkat cells control group and RS4;11 cells control group were cultured synchronously using RPMI1640 medium.The cell growth curves of the four groups were analyzed by CCK-8.The cell apoptosis of the four groups were analyzed by Annexin Ⅴ-allophycocyanin （APC）/7-aminoactinomycin D （7-AAD） double labeling flow cytometry.The cell cycle changes of the four groups were analyzed by flow cytometry.The mRNA expressions levels of B cell lymphoma/ leukemia-2 associated Ⅹ protein （Bax） gene,B cell lymphoma/ leukemia （Bcl）-2 gene,X-linked inhibitor of apoptosis protein （XIAP） gene,cyclin-dependent kinase （CDK） 1 gene and cysteine-aspartic proteases （caspase）-3 gene of the four groups were determined by real-time PCR.Results ①b-AP15 significantly inhibited the growth of J urkat cells and RS4;11 cells,and both the inhibitory effect were in a time-and dose-dependent manner.The IC50 values of J urkat cells were（1.52 ± 0.35）μmol/L and（0.54-±-0.01）μmol/L after culturing with b-AP15 for 24 h and 48 h.The IC50 values of RS4;11 cells were （0.97±0.02）μmol/L and（0.08± 0.03）μmol/L after culturing with b-AP15 for 24 h and 48 h.②The apoptosis rates of Jurkat cells were significantly higher than the control groups after cultured with 1.5 μmol/L b-AP15 （P＜0.001）.The apoptosis rates of RS4;11 cells significantly higher than the control groups

关 键 词：蛋白酶抑制药 细胞增殖 细胞凋亡 细胞周期 JURKAT细胞 RS4 11细胞 b-AP15 

分 类 号：R3416[医药卫生—基础医学]