异藤黄酚对急性淋巴细胞白血病Jurkat细胞增殖和凋亡的影响及机制  

Effectsof isogarcinol on proliferation and apoptosis of Jurkat cells in acute lymphoblastic leukemia and its mechanism

作　　者：刘琴 杨坤[1,2,3] 牛振鹏 韦学耐[1,2,3] 宋晶睿 饶青 黄裕兵 苑春茂 李艳梅 LIU Qin;YANG Kun;NIU Zhenpeng;WEI Xuenai;SONG Jingrui;RAO Qing;HUANG Yubing;YUAN Chunmao;LI Yanmei(School of Pharmacy,Guizhou Medical University,Guiyang 550004,Guizhou,China;Guizhou Natural Products Research Center,Guiyang 550014,Guizhou,China;Provincial and Ministerial State Key Laboratory for Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550014,Guizhou,China;School of Basic Medicine,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学药学院,贵州贵阳550004 [2]贵州省天然产物研究中心,贵州贵阳550014 [3]贵州医科大学、省部共建药用植物功效与利用国家重点实验室,贵州贵阳550014 [4]贵州医科大学基础医学院,贵州贵阳550004

出　　处：《贵州医科大学学报》2023年第11期1273-1281,1291,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81960546);贵州省科技计划项目(黔科合平台人才〔2020〕5008)。

摘　　要：目的探讨异藤黄酚(ISO)对急性淋巴细胞白血病(ALL)Jurkat细胞增殖和凋亡的影响及机制。方法人ALL Jurkat细胞和人正常肝细胞HL-7702分别培养至对数生长期,0[二甲基亚砜(DMSO)]、10、15、20及25μmol/L ISO处理2种细胞,采用噻唑蓝(MTT)法检测各组细胞处理后24、48及72 h的光密度(OD)并计算细胞存活率;取对数生长期Jurkat细胞,分别用0(DMSO)、10、15及20μmol/L ISO处理,采用流式细胞术检测各浓度ISO组Jurkat细胞24、48 h的凋亡率,采用Hoechst 33258染色法验证各浓度ISO组Jurkat细胞24 h的凋亡率,采用线粒体膜电位(MMP)检测试剂盒(JC-1)检测各浓度ISO组Jurkat细胞24 h的MMP,采用RNA高通量测序(RNA-seq)分析ISO组Jurkat细胞24 h的细胞内基因转录水平,采用Western blot检测各浓度ISO组Jurkat细胞24 h时凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(Caspase3)、半胱氨酸天冬氨酸蛋白酶9(Caspase9)、剪切的Caspase3(Cleaved-caspase3)、剪切的Caspase9(Cleaved-caspase9)、凋亡蛋白酶活化因子-1(Apaf-1)、细胞色素C(Cytc)、蛋白激酶B(Akt)、磷脂酰肌醇-3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、重组人BH3结构域凋亡诱导蛋白(Bid)、截断的Bid(t-Bid)、磷酸化的信号转导和转录激活因子-3(p-STAT3)、Janus激酶2(JAK2)、磷酸化JAK2(p-JAK2)、多聚腺苷二磷酸核糖聚合酶(PARP)、剪切的PARP(Cleaved-PARP)、丝裂原活化蛋白激酶p38(p38)蛋白的表达。结果MTT检测结果显示,与DMSO组相比,10、15、20及25μmol/L ISO组Jurkat细胞活力下降(P<0.05),且具有时间依赖性;流式细胞术结果显示,与DMSO组相比,10、15及20μmol/L ISO组Jurkat细胞凋亡率升高(P<0.05),且具有时间依赖性;Western blot结果显示,与DMSO组相比,10、15及20μmol/L ISO处理Jurkat细胞24 h后,Cleaved-caspase3、Cleaved-caspase9、Cleaved-PARP、t-Bid、Apaf-1及Cytc的表达上调(P<0.05),p-JAK2、p-STAT3及c-Myc的表达下调(P<0.05);RNAseq分析结果,与DMSO组相比,15μmol/L ISO处理Jurkat细�Objective To investigate the effects and mechanism of isogarcinol(ISO)on the proliferation and apoptosis of Jurkat cells in acute lymphoblastic leukemia(ALL).Methods Human ALL Jurkat cells and human normal hepatocytes HL-7702 were cultured to the logarithmic growth stage and then exposed to 0(DMSO),10,15,20,and 25μmol/L ISO.The optical density(OD)of each cell group was detected by thiazole blue(MTT)assay at 24,48,and 72 h,and the cell viability was calculated.Jurkat cells at logarithmic growth stage were chosen and exposed to 0(DMSO),10,15,and 20μmol/L ISO.Additionally,the apoptosis rate of Jurkat cells in each concentration of ISO group was examined by flow cytometry for 24 and 48 h,and 24 h apoptosis rate of Jurkat cells was verified by Hoechst 33258 staining.The mitochondrial membrane potential(MMP)of Jurkat cells in each concentration of ISO group was also analyzed by mitochondrial membrane potential detection kit(JC-1).RNA high-throughput sequencing(RNA-seq)was used to analyze the intracellular gene transcription level of 24 h Jurkat cells of ISO groups.Western blot was adopted to detect apoptosis-associated proteins expression of 24 h Jurkat cells of ISO groups with varied concentrations:cysteine aspartate protease 3(Caspase3),cysteine aspartate protease 9(Caspase9),sclipped cysteine aspartate protease 3(Cleaved-caspase3),cleavage cysteine aspartate protease 9(Cleaved-caspase9),Apoptotic protease activating factor-1(Apaf-1),cytochrome C(Cytc),protein kinase B(Akt),phosphatidylinositol-3-kinase(PI3K),phosphorylated PI3K(p-PI3K),recombinant human BH3 domain apoptosis-inducing protein(Bid),truncated recombinant human BH3 domain apoptosis-inducing protein(t-Bid),phosphorylated signal transduction and transcription activator-3(p-STAT3),Janus kinase 2(JAK2),phosphorylated JAK2(p-JAK2),polyadenosine diphosphate ribose polymerase(PARP),cleavage polyadenosine diphosphate ribose polymerase(Cleaved-PARP),and mitogen-activated protein kinase p38(p38).Results The MTT assay revealed that Jurkat cell viability was decre

关 键 词：细胞凋亡 细胞增殖 JURKAT细胞 异藤黄酚 急性淋巴细胞白血病 线粒体膜电位 

分 类 号：R733.7[医药卫生—肿瘤]