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
26S단백매억제제b-AP15대Jurkat급RS4;11세포증식、조망적영향급조망궤제적연구
Role of the 26S proteasome inhibitor b-AP15 on Jurkat cells and RS4;11 cells
  • 万方数据
    国际输血及血液学杂志 국제수혈급혈액학잡지
    INTERNATIONAL JOURNAL OF BLOOD TRANSFUSION AND HEMATOLOGY
    2015年 3期 185-191 ,共7页

    周俊%曹江%孟凡静%冯浩%徐开林

    주준%조강%맹범정%풍호%서개림

    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 蛋白酶抑製藥%細胞增殖%細胞凋亡%細胞週期%Jurkat細胞%RS4%11細胞%b-AP15
    단백매억제약%세포증식%세포조망%세포주기%Jurkat세포%RS4%11세포%b-AP15
    Protease inhibitor%Cell proliferation%Apoptosis%Cell cycle%Jurkat cells%RS4%11 cells%b-AP15
    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细胞周期明显被阻滞于G2/M期,且差异有统计学意义(t=10.672,P＜0.05);同时伴S期细胞比例减低,且差异亦有统计学意义(t=19.053,P＜0.05).与RS4;11细胞对照组相比,使用浓度为1.0 μmol/L b-AP15处理RS4;11细胞实验组24 h后,RS4;11细胞周期亦明显被阻滞于G2/M期,且差异有统计学意义(t=13.643,P＜0.05);同时伴S期细胞比例减低,且差异亦有统计学意义(t=6.992,P＜0.05).④使用b-AP15处理Jurkat细胞与RS4;11细胞24 h后,荧光定量PCR检测结果显示,凋亡相关基因Bax、caspase-3 mRNA相对表达水平增高,Bcl-2、XIAP mRNA相对表达水平减低,同时伴细胞周期相关基因CDK1 mRNA相对表达水平减低,与各自细胞对照组相比,差异均有统计学意义(P＜0.05);且RS4;11细胞实验组caspase-3 mRNA相对表达水平较Jurkat细胞实验组相比,增高程度更为显著,且差异有统计学意义(P＜0.05).结论 b-AP15能有效抑制Jurkat细胞与RS4;11细胞增殖,并促进其细胞凋亡,使细胞周期阻滞于G2/M期,其促凋亡机制与上调Bax与caspase-3基因表达水平、下调Bcl-2与XIAP基因表达水平相关,细胞周期G2/M期阻滞与CDK1基因表达水平减低相关.
    목적 탐토26S단백매억제제b-AP15대Jurkat세포여RS4;11세포증식급세포조망적영향,병분석기작용궤제.방법 무균조건하대Jurkat세포여RS4;11세포진행배양,근거유무b-AP15처리,장기분위4조:①Jurkat세포실험조;②Jurkat세포대조조;③RS4;11세포실험조;④RS4;11세포대조조.기중,Jurkat세포실험조여RS4;11세포실험조채용농도분별위0.05,0.10,0.50,1.00,5.00 μmol/L b-AP15처리24,48 h;Jurkat세포대조조여RS4;11세포대조조채용RPMI 1640배양기진행동보배양.채용CCK-8법회제4조세포증식곡선;채용Annexin Ⅴ-별조람단백(APC)/7-안기방선균소D(7-AAD)쌍표기류식세포술검사4조세포조망정황;채용류식세포술검측세포4조세포주기분포정황;채용실시정량PCR검측4조세포B세포림파류/백혈병-2상관Ⅹ단백(Bax)기인、B세포림파류/백혈병(Bcl)-2기인、Ⅹ련쇄조망억제단백(XIAP)、주기소의뢰성격매(CDK)1기인급반광안산천동안산단백매(caspase)-3기인mRNA표체수평.결과 ①b-AP15대Jurkat세포여RS4;11세포증식균구유억제작용,차해억제효응정시간여제량의뢰성;Jurkat세포경b-AP15처리24,48 h후,반수억제농도(IC50)분별위(1.52±0.35)μmol/L여(0.54±0.01) μmol/L,RS4;11세포경b-AP15처리24,48 h후IC50분별위(0.97±0.02)μmol/L여(0.08±0.03)μmol/L.②사용농도위1.5 μmol/L b-AP15처리Jurkat세포실험조후,기여Jurkat세포대조조상비세포조망솔현저증고,차차이유통계학의의(P＜0.001);사용농도위1.0 μmol/Lb AP15처리RS4;11세포실험조후,기여RS4;11세포대조조상비세포조망솔역현저증가,차차이역유통계학의의(P＜0.001);차Jurkat세포여RS4;11세포경b-AP15처리48 h후,여처리24 h후자상비세포조망솔증고,차차이균유통계학의의(t=11.887,7.449;P＜0.001).③여Jurkat세포대조조상비,사용농도위1.5 μmol/L b-AP15처리Jurkat세포실험조24 h후,Jurkat세포주기명현피조체우G2/M기,차차이유통계학의의(t=10.672,P＜0.05);동시반S기세포비례감저,차차이역유통계학의의(t=19.053,P＜0.05).여RS4;11세포대조조상비,사용농도위1.0 μmol/L b-AP15처리RS4;11세포실험조24 h후,RS4;11세포주기역명현피조체우G2/M기,차차이유통계학의의(t=13.643,P＜0.05);동시반S기세포비례감저,차차이역유통계학의의(t=6.992,P＜0.05).④사용b-AP15처리Jurkat세포여RS4;11세포24 h후,형광정량PCR검측결과현시,조망상관기인Bax、caspase-3 mRNA상대표체수평증고,Bcl-2、XIAP mRNA상대표체수평감저,동시반세포주기상관기인CDK1 mRNA상대표체수평감저,여각자세포대조조상비,차이균유통계학의의(P＜0.05);차RS4;11세포실험조caspase-3 mRNA상대표체수평교Jurkat세포실험조상비,증고정도경위현저,차차이유통계학의의(P＜0.05).결론 b-AP15능유효억제Jurkat세포여RS4;11세포증식,병촉진기세포조망,사세포주기조체우G2/M기,기촉조망궤제여상조Bax여caspase-3기인표체수평、하조Bcl-2여XIAP기인표체수평상관,세포주기G2/M기조체여CDK1기인표체수평감저상관.
    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 after cultured with 1.0 μmol/L b-AP15 (P＜0.001).Meanwhile,The apoptosis rates of both Jurkat cells and RS4;11 cells cultured with b-AP15 for 48 h were significantly higher than these for 24 h (t=11.887,7.449;P＜0.001).③ Compared with Jurkat cells control group,cell cycle of Jurkat cells experimental group cultured with 1.5 μmol/L b-AP15 for 24 h was arrested at G2/M period (t=10.672,P＜0.05).At the same time,the proportion of S phase cells were reduced significantly (t=19.053,P＜0.05).Compared with RS4;11 cells control group,cell cycle of RS4;11 cells experimental group cultured with 1.0 μmol/L b-AP15 for 24 h was also arrested at G2/M period (t=13.643,P＜0.05).and the proportion of S phase cells were also reduced significantly(t=6.992,P＜0.05).④For Jurkat cells experimental group and RS4;11 cells experimental group,the realtime PCR assay revealed that the relative mRNA expression levels of Bax gene,caspase-3 gene were increased significantly,while the relative mRNA expression levels of Bcl-2 gene,XIAP gene were reduced significantly.Meanwhile,mRNA expression level of the cell cycle related gene CDK1 was reduced significantly compared with their own control groups (P＜0.05).The mRNA expression level of caspase-3 gene in RS4;11 cells experimental group increased more significantly than that of Jurkat cells experimental group (P＜0.05).Conclusion b-AP15 can inhibit proliferation and induce apoptosis of Jurkat cells and RS4;11 cells,and the cell cycle of the two cells were both arrested at G2/M period.The mechanism of b-AP15 promoting apoptosis may be related with up-regulating the expression levels of Bax gene,caspase-3 gene and down-regulating the expression levels of Bcl-2 gene,XIAP gene.And the cell cycle arrested at G2/M period may be associated with down-regulating the expression levels of CDK1 gene.
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