黑骨藤对高表达表皮生长因子受体突变体Ⅲ的人胶质母细胞瘤细胞DKMG的影响及机制  

Effect of periploca forrestii schltr on human glioblastoma cell DKMG with high expression of epidermal growth factor receptor variantⅢand its mechanism

作　　者：唐银彤 官志忠[3] 陆定艳 陈帅帅 吴忠秀 曹陶涛 郭玮钰 刘亭 TANG Yingtong;GUAN Zhizhong;LU Dingyan;CHEN Shuaishuai;WU Zhongxiu;CHAO Taotao;GUO Weiyu;LIU Ting(School of Pharmacy,Guizhou Medical University,Guiyang 550004,Guizhou,China;Guizhou Provincial Key Laboratory of Pharmaceutics&State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education&Key Laboratory of Medical Molecular Biology of Guizhou Province,Guizhou Medical University,Guiyang 550001,Guizhou,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,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]贵州医科大学贵州省药物制剂重点实验室&省部共建药用植物功效与利用国家重点实验室,贵州贵阳550004 [3]贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省医学分子生物学重点实验室,贵州贵阳550001 [4]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵州贵阳550004

出　　处：《贵州医科大学学报》2023年第10期1137-1144,共8页Journal of Guizhou Medical University

基　　金：NSFC-喀斯特联合基金项目(U1812403);贵州省科技计划项目(黔科合基础-ZK〔2022〕重点037);贵州省优秀青年科技人才项目(黔科合平台人才〔2021〕5619号);贵州省普通高等学校科技拔尖人才项目(黔教合KY字〔2021〕033)。

摘　　要：目的探讨黑骨藤对高表达表皮生长因子(EGF)受体突变体Ⅲ(EGFRvⅢ)的人胶质母细胞瘤细胞DKMG的影响及其机制。方法取对数生长期的DKMG和不表达EGFRvⅢ的人脑星形胶质母细胞瘤细胞U87MG,各自分为空白(Con)组(DMEM培养液)、低浓度黑骨藤组(100 mg/L黑骨藤培养液)、中浓度黑骨藤组(200 mg/L黑骨藤培养液)及高浓度黑骨藤组(400 mg/L黑骨藤培养液),处理细胞24 h,采用细胞增殖与毒力检测试剂盒(MTS)检测各组DKMG、U87MG细胞的存活率,采用流式细胞术检测各组DKMG细胞凋亡率;实时荧光定量多聚核苷酸链式反应(qRT-PCR)检测各组DKMG细胞中EGFRvⅢ、B淋巴细胞瘤-2基因(Bcl-2)及BCL2-Associated X(Bax)的信使核糖核酸(mRNA)表达,采用蛋白免疫印迹法(Western blot)检测各组DKMG细胞中EGFRvⅢ、Bcl-2、Bax、半胱氨酸蛋酶家族(Caspase-3、Cleaved caspase-3)蛋白的表达。结果MTS检测显示,与Con组相比,各浓度黑骨藤组DKMG细胞存活率明显降低(P<0.05),各浓度黑骨藤组U87MG细胞存活率比较、差异无统计学意义(P>0.05);流式细胞术结果表明,与Con组比较,各浓度黑骨藤组DKMG细胞凋亡率明显增加(P<0.05);qRT-PCR结果表明,与Con组比较,各浓度黑骨藤组DKMG细胞中EGFRvⅢ和Bcl-2 mRNA表达降低(P<0.05),Bax表达无变化(P>0.05);Western blot结果显示,各浓度黑骨藤组DKMG细胞中EGFRvⅢ、Bcl-2及Caspase-3蛋白表达下降(P<0.05),Bax、Cleaved caspase-3蛋白表达升高(P<0.05)。结论黑骨藤可诱发DKMG细胞凋亡,产生细胞杀伤作用,其机制可能与下调EGFRvⅢ、Bcl-2、Caspase-3表达及上调Bax、Cleaved caspase-3表达有关。Objective To investigate the toxic effect and mechanism of Periploca forrestii Schltr on human glioblastoma cell lines(DKMG)with high expression of epidermal growth factor receptor variantⅢ(EGFRvⅢ).Methods DKMG from logarithmic growth phase and U87MG from human astrocytoma cells without EGFRvⅢexpression were obtained and divided into control group(DMEM culture medium),low concentration Periploca forrestii Schltr group(100 mg/L Periploca forrestii Schltr culture medium),medium concentration Periploca forrestii Schltr group(200 mg/L Periploca forrestii Schltr culture medium),and high concentration Periploca forrestii Schltr group(400 mg/L Periploca forrestii Schltr culture medium).The cells were treated for 24 hours and the cell viability of DKMG and U87MG cells in each group was detected by MTS Cell Proliferation and Virulence Detection Kit(MTS).The apoptosis rate of DKMG cells in each group was detected by flow cytometry.Real-time fluorescence quantitative polynucleotide chain reaction(qRT-PCR)was used to detect the expression of EGFRvⅢ,B lymphocyte tumor-2 gene(Bcl-2),and BCL2-AssociatedX(Bax)in DKMG cells of each group.Western blot was used to detect the expression of EGFRvⅢ,Bcl-2,Bax,cysteine methionase family(Caspase-3,Cleaved caspase-3)in DKMG cells of each group.Results The results of MTS showed that compared with the control group,the cell viability of DKMG cells in each concentration of Periploca forrestii Schltr group decreased significantly(P<0.01),whereas there was no significant difference in the cell viability of U87MG cells in each concentration group(P>0.05).The results of flow cytometry indicated that the apoptosis rate of DKMG cells in each concentration of Periploca forrestii Schltr group was significantly increased compared with the control group(P<0.01).The results of qRT-PCR revealed that compared with the control group,the expression of EGFRvⅢand Bcl-2 mRNA in DKMG cells of all concentrations of Periploca forrestii Schltr group decreased(P<0.05),but the expression of Bax remained

关 键 词：细胞凋亡 黑骨藤 DKMG细胞 U87MG细胞 表皮生长因子受体突变体Ⅲ 细胞毒性 

分 类 号：R965[医药卫生—药理学]