汉防己乙素衍生物LYY-47对三阴性乳腺癌细胞及其多倍体巨瘤细胞增殖、凋亡的作用和机制  

Effect and mechanism of tetrandrine derivative LYY-47 on proliferation and apoptosis of triple negative breast cancer cells and their polyploid giant cancer cells

作　　者：余晓静 张望明 贺天辉 刘小花 艾海锋 安丽君 杨留启 潘卫东 刘杰麟 YU Xiaojing;ZHANG Wangming;HE Tianhui;LIU Xiaohua;AI Haifeng;AN Lijun;YANG Liuqi;PAN Weidong;LIU Jielin(Department of Immunology,School of Basic Medical Science,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Clinical Laboratory,Jiujiang University Hospital,Jiujiang 332000,Jiangxi,China;Department of Clinical Laboratory,Dejiang Ethenic Hospital of Traditional Chinese Medicine,Tongren 565200,Guizhou,China;School of Pharmacy,Guizhou University,Guiyang 550025,Guizhou,China;State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550014,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院免疫学教研室,贵州贵阳550025 [2]九江学院附属医院检验科,江西九江332000 [3]德江县民族中医院检验科,贵州铜仁565200 [4]贵州大学药学院,贵州贵阳550025 [5]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

出　　处：《贵州医科大学学报》2024年第3期313-328,共16页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82260458);贵州省科技支撑项目(黔科合支撑[2020]4Y120);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开放课题项目(FAMP202110K);国基培育项目(21NSFCP27)

摘　　要：目的探讨汉防己乙素衍生物LYY-47对三阴性乳腺癌(TNBC)细胞及其多倍体巨瘤细胞(PGCCs)增殖、凋亡的作用和机制。方法取TNBC MDA-MB-231细胞和MDA-MB-436细胞培养至对数生长期,诱导形成PGCCs,培养35 d,采用苏木素-伊红(H&E)染色观察不同培养时间时2种细胞的PGCCs形态学特征并进行计数;收集MDA-MB-231细胞、PGCCs及其子代细胞,采用流式细胞仪检测分析细胞周期,采用Western blot检测周期相关蛋白[细胞周期蛋白依赖性激酶1(CDK1)和细胞周期蛋白B1(CyclinB1)]、干性相关蛋白[乙醛脱氢酶1A1(ALDH1A1)、白细胞分化抗原44(CD44)及白细胞分化抗原133(CD133)]、DNA损伤修复相关蛋白[布卢姆(BLM)、Rad51及乳腺癌易感基因1(BRCA1)]及凋亡相关蛋白[BCL2-相关X蛋白(Bax)、B淋巴细胞瘤蛋白-2(Bcl-2)、裂解凋亡蛋白酶-3(cleaved Caspase-3)及裂解凋亡蛋白酶-8(cleaved Caspase-8)]的表达,采用噻唑蓝(MTT)法和克隆形成实验检测细胞活力和细胞集落数,采用细胞免疫荧光实验检测磷酸化组蛋白2AX(γ-H2AX)的表达;采用荧光偏振实验检测BLM DNA解旋酶的活性;收集对数生长期MDA-MB-231细胞,分为对照组(同等体积的完全培养基)、紫杉醇(PTX)组(500 nmol/L PTX)、3-CF 3,4-F-苯基类似物(ML216)组(3μmol/L ML216)及ML216+PTX组(500 nmol/L PTX和3μmol/L ML216),采用Image J软件计数各组细胞数;收集对数生长期MDA-MB-231细胞及其PGCCs子代细胞,分为对照组(0.00μmol/L)、LYY-47给药组(2.50μmol/L、5.00μmol/L及6.50μmol/L),采用流式细胞仪检测上述各组细胞的凋亡情况;6周龄雌性无特定病原体(SPF)级无胸腺BALB/c裸鼠12只,皮下分别注射5×10^(6)个MDA-MB-231细胞及其PGCCs子代细胞,每隔3天测量1次肿瘤体积,连续29 d,处死裸鼠剥离肿瘤、称重,取肿瘤组织制作切片,采用H&E染色和免疫组织化学染色观察细胞形态和检测BLM、Ki-67的表达。结果与TNBC MDA-MB-231和MDA-MB-436细胞相比,PTX诱导的PGCCs出现�Objective To explore the effects and mechanism of tetrandrine derivative LYY-47 on the proliferation and apoptosis of triple negative breast cancer(TNBC)cells and polyploid giant cancer cells(PGCCs)with budding.Methods TNBC MDA-MB-231 cells and MDA-MB-436 cells were cultured to logarithmic growth phase to induce PGCCs.After 35 days of culture,H&E staining was used to observe the morphological characteristics of PGCCs of the two types at different culture time,and the PGCCs were counted.MDA-MB-231 cells,PGCCs and their PGCCs with budding were collected and analyzed for cell cycle by Flow cytometry.Western blot was used to detect the expression of cyclin-related proteins[cyclin dependent kinase 1(CDK1)and Cell cycle proteinB1(CyclinB1)],stemness related protein[acetaldehyde dehydrogenase1A1(ALDH1A1),cluster of differentiation 44(CD44)and cluster of differentiation 133(CD133)],DNA damage repair related protein[Bloom(BLM),(Rad51)and breast cancer susceptibility gene 1(BRCA1)]and apoptosis-related proteins[Bcl-2-associated X protein(Bax),B-cell lymphoma protein2(Bcl-2),cleaved Caspase-3,and cleaved Caspase-8].Methyl thiazol tetrazolium(MTT)assay and colony formation assay were used to detect cell viability and colony number.Immunofluorescence assay was used to detect the expression of phosphorylated histone 2AX(γ-H2AX).The activity of BLM DNA helicase was detected by fluorescence polarization assay.MDA-MB-231 cells in logarithmic growth phase were collected.They were divided into the control group(the same volume of complete medium),PTX group(500 nmol/L PTX),3-CF^(3),4-F-phenyl analog(ML216)group(3μmol/L ML216)and ML216+PTX group(500 nmol/L PTX and 3μmol/L ML216).Image J software was used to count the number of cells in each group.Logarithmic growth MDA-MB-231 cells and PGCCs with budding were collected and divided into the control group(0.00μmol/L)and LYY-47 treatment group(2.50μmol/L,5.00μmol/L,and 6.50μmol/L).The apoptosis of the above cells was detected by flow cytometry.Twelve 6-week-old female nude mice o

关 键 词：乳腺肿瘤 DNA解旋酶类 细胞增殖 细胞凋亡 汉防己乙素衍生物 多倍体巨瘤细胞 BLM DNA解旋酶 

分 类 号：R737.9[医药卫生—肿瘤]