摘要
目的 探讨4型含Bromo结构域蛋白（BRD4）拮抗剂对普通型急性B淋巴细胞白血病（B-ALL）患者原代白血病细胞的作用及可能机制.方法 采用流式细胞术分选14例普通型B-ALL患者（Ph＋ B-ALL4例,Ph-B-ALL 10例）白血病细胞,在模拟骨髓微环境条件下进行短期培养;给予BRD4拮抗剂GSK525762A处理后,采用CCK-8法检测细胞增殖抑制率,用Annexin V/7-AAD法检测细胞凋亡率,荧光定量PCR法检测c-MYC、CDK6、Bcl-2 mRNA表达水平,Western blot法检测c-MYC、CDK6、Bcl-2蛋白的表达.结果 GSK525762A对14例普通型B-ALL患者原代白血病细胞均有抑制增殖作用,且呈剂量依赖性,其中位IC50值为256.25 （90.64～1 378.39） nmol/L.500、1 000、2 500 nmol/LGSK525762A作用后中位细胞凋亡率分别为45.17％ （9.38％～70.91％）、66.02％ （24.36％～96.34％）、89.29％（39.29％～99.37％）.GSK525762A对ph＋与Ph-B-ALL细胞具有类似的作用,但对Ph＋B-ALL细胞抑制增殖和诱导凋亡作用均弱于Ph-B-ALL细胞.1 000 nmol/L GSK525762A处理白血病细胞24、48 h后,c-MYC、CDK6和Bcl-2 mRNA表达水平下降,其中对ph＋和Ph-B-ALL细胞c-MYC、CDK6mRNA的下调作用差异无统计学意义,而对Ph＋ B-ALL细胞Bcl-2 mRNA表达水平的下调弱于对Ph-B-ALL细胞;GSK525762A处理后白血病细胞c-MYC、CDK6、Bcl-2蛋白表达水平均下调.结论 GSK525762A可抑制普通型B-ALL原代细胞的增殖,并促进其凋亡,且对Ph＋ ALL细胞在体外具有一定作用.该作用可能通过下调c-MYC、CDK6和Bcl-2而实现.
Objective To investigate the effects of bromodomain-containing protein 4 （BRD4） inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia （common B-ALL）cells from adult patients,then to further explore the possible mechanisms.Methods Purified leukemia cells from 14 common B-ALL adult patients （4 Ph＋ and 10 Ph-cases）were obtained by flow cytometry sorting,and maintained in a mimic bone marrow microenvironment culture system for short-term culture.Leukemia cells were treated with various concentrations of GSK525762A.The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by Annexin V/7-AAD staining using flow cytometry.The transcripts of c-MYC,CDK6 and Bcl-2 were detected by quantitative RT-PCR,and the expression of c-MYC,CDK6 and Bcl-2 proteins were detected via Western blot.Results GSK525762A could inhibit the proliferation of leukemia cells from all 14 conmon B-ALL patients in a dose-dependent manner,the median value of IC50 was 256.25 （90.64-1 378.39） nmol/L.GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner,the median apoptosis rates respectively were 45.17% （9.38%-70.91%）,66.02% （24.36%-96.34%） and 89.29% （39.29%-99.37%） after treated by 500,1 000 and 2 500 nmol/L GSK525762A.GSK525762A has a similar effect on Ph＋ ALL and Ph-B-ALL,but theeffect of proliferation inhibition and apoptosis enhancement on Ph＋ B-ALL is weaker than that on Ph-B-ALL.Compared with vehicle control group,the levels of c-MYC,Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A,and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph＋ and Ph-B-ALL;however,the inhibitory effect on Bcl-2 transcription was weaker in Ph＋ B-ALL cells than that in Ph-B-ALL cells.Moreover,c-MYC,Bcl-2 and CDK6 protein levels decreased in GS
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2015年第7期563-569,共7页
Chinese Journal of Hematology
基金
国家自然科学基金（81000210、81471580、81300399、81270637）
江苏省自然科学基金（BK20141138）
江苏省临床医学科技专项（BL2013010）
