摘要
目的探讨第二个线粒体衍生的半胱氨酸蛋白酶激动剂小分子(Smac)类似物LCL161联合天冬氨酸蛋白水解酶(caspase)抑制剂Z-VAD-FMK诱导细胞发生坏死性凋亡对人乳腺癌细胞MCF-7和MDA-MB-231增殖的影响。方法体外培养MCF-7、MDA-MB-231细胞株。噻唑蓝(MTT)法检测2种细胞株空白对照组、Z-VAD-FMK组(10 mmol/L)、LCL161(1μmol/L)组及LCL161+Z-VAD-FMK组存活率,并应用透视电子显微镜观察各组细胞凋亡亚微结构。Nec-1预处理后,MTT法检测2种细胞株空白对照组、Nec-1组(20 mmol/L),LCL161(1μmol/L)组及LCL161+Nec-1组细胞存活率。Annexin V-FITC/PI双染法检测空白对照组、LCL161组、Z-VAD组、Nec-1组、LCL161+Z-VAD组、LCL161+Nec-1组、LCL161+Z-VAD+Nec-1组细胞凋亡率。裸鼠成瘤实验观察空白对照组、LCL161组、LCL161+Z-VAD-FMK组裸鼠活体内MCF-7细胞株移植瘤体积和质量。结果(1)MTT法检测结果显示,LCL161+Z-VAD-FMK组的MDA-MB-231和MCF-7细胞株的存活率均低于其他各组,差异均有统计学意义(P值均<0.01)。透视电子显微镜观察到LCL161+Z-VAD-FMK组细胞具有凋亡和坏死共同存在的特征性形态学改变。(2)Nec-1预处理后,MTT法检测结果显示,与空白对照组及Nec-1组相比较,LCL161组和Nec-1+LCL161组的MDA-MB-231和MCF-7细胞的OD值明显降低,表明其细胞存活率较低,差异均有统计学意义(P值均<0.05);但LCL-161组与Nec-1+LCL161组比较,MDA-MB-231和MCF-7细胞存活率差异均无统计学意义(P值均>0.05)。(3)Annexin V-FITC/PI双染法结果显示,与其他各组相比,LCL161+Z-VAD-FMK组和LCL161组两种细胞株凋亡率明显增高,LCL161+Z-VAD-FMK组亦高于LCL161组,差异均有统计学意义(P值均<0.01)。(4)15只裸鼠成功建立MCF-7细胞裸鼠成瘤模型,给药后第20天时LCL161组和LCL161+Z-VAD-FMK组肿瘤体积和质量均低于空白对照组,差异均有统计学意义(P值均<0.05),而LCL161组与LCL161+Z-VAD-FMK组的肿瘤体积和质量差异均无统计学意义(P值�
Objective To investigate the effect of a second mitochondria-derived cysteine protease agonist small molecule(Smac)analog,LCL161,combined with aspartate protein hydrolase(caspase)inhibitor Z-VAD-FMK to induce necroptosis on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.Methods MCF-7 and MDA-MB-231 cell lines were cultured in vitro,and the survival rates of the blank control,Z-VAD-FMK(10 mmol/L),LCL161(1μmol/L)and LCL161+Z-VAD-FMK groups of the two cell lines were examined by MTT,and the apoptotic submicrostructures were observed by fluoroscopic electron microscopy.dec-1 After Nec-1 pretreatment,the survival rates of the blank control,Nec-1(20 mmol/L),LCL161(1μmol/L)and LCL161+Nec-1 groups were detected by MTT assay.Annexin V-FITC/PI double staining method to detect cell apoptosis in blank control group,LCL161 group,Z-VAD-FMK group,LCL161+Nec-1 group and LCL161+Z-VAD+Nec-1 group.Nude mice tumorigenesis assay was performed to observe the volume and quality of transplanted MCF-7 cell lines in vivo in the blank control group,LCL161 group and LCL161+Z-VAD-FMK group.Results(1)The results of MTT assay showed that the survival rates of MDA-MB-231 and MCF-7 cell lines in the LCL161+Z-VAD-FMK group were lower than those in all other groups,and the difference were statistically significant(all P values<0.01).The cells in the LCL161+Z-VAD-FMK group were observed by fluoroscopic electron microscopy to have characteristic morphological changes of apoptosis and necrosis together.(2)After Nec-1 pretreatment,the results of MTT assay showed that the survival rate of MDA-MB-231 and MCF-7 cells in the LCL161 group and the Nec-1+LCL161 group was significantly reduced compared with the blank control group and the Nec-1 group,which showed that the cell survival rate was low,and the differences were statistically significant(all P values<0.05),but the difference between the LCL-161 and Nec-1+LCL161 groups were not statistically significant(all P values>0.05).(3)The results of Annexin V-FITC/PI double-staining met
作者
金功圣
乔梦祥
陈延松
张晓静
刘先富
Jing Gongsheng;Qiao Mengxiang;Chen Yansong;Zhang Xiaojing;Liu Xianfu(Department of Oncology Surgery,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China)
出处
《中华解剖与临床杂志》
2021年第5期578-584,共7页
Chinese Journal of Anatomy and Clinics
基金
安徽高校自然科学研究重点项目(KJ2017A245)。
