楝酰胺对肝癌细胞的促凋亡作用及其机制  

Effect of rocaglamide on apoptosis of hepatocellular carcinoma cells and its mechanism

作　　者：楚蕾蕾 高国伟[2] 杨桢桢 杨留勤[2] 武莉萍[2] CHU Leilei;GAO Guowei;YANG Zhenzhen;YANG Liuqin;WU Liping(Department of Radiotherapy,The Fourth Clinical College of Xinxiang Medical University,Xinxiang 453000,China)

机构地区：[1]新乡医学院第四临床学院,河南新乡453000 [2]新乡市中心医院放疗科

出　　处：《山东医药》2022年第6期6-11,共6页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81572939)。

摘　　要：目的观察加入楝酰胺(Rocaglamide,Roc-A)培养的人肝癌细胞株HepG2凋亡情况,并探讨其可能作用机制。方法①加入不同浓度Roc-A培养的HepG2细胞凋亡情况观察:取对数生长期HepG2细胞,分为5组,分别加入0(空白对照)、25、50、100、200 nmol/L的Roc-A溶液,培养24 h时取各组细胞,采用YF488-AnnexinV/PI荧光双染法观察细胞凋亡情况。②加入不同浓度Roc-A培养的HepG2细胞已糖激酶Ⅱ(HK2)、抑凋亡因子类似物(BCL2L1)mRNA检测:取对数生长期HepG2细胞,分为2组,分别加入0、100 nmol/L的Roc-A溶液,培养24 h时采用RT-qPCR法检测细胞HK2及BCL2L1 mRNA。③加入不同浓度Roc-A培养的HepG2细胞HK2、BCL2L1蛋白检测:取适量HepG2细胞,分为3组,分别加入0、100、200 nmol/L的Roc-A,培养24 h采用蛋白组学质谱分析法检测细胞HK2、BCL2L1蛋白。取对数生长期HepG2细胞分为5组,分别加入0、25、50、100、200 nmol/L的Roc-A溶液,培养24 h时采用WesternBlotting法检测HK2、BCL2L1蛋白。④癌组织HK2表达与肝癌患者的生存时间关系分析:采用KaplanMeierPlotter癌症数据库分析癌组织HK2表达与肝癌患者生存时间的关系。结果加入0、25、50、100、200 nmol/L的Roc-A溶液HepG2细胞凋亡率分别为0.83%±0.21%、9.46%±2.34%、24.68%±4.22%、39.60%±2.16%及58.63%±1.62%,组间两两比较,Р<0.05。随Roc-A溶液浓度增加,细胞凋亡率也随之增加。与空白对照比较,培养24 h时加入100 nmol/L Roc-A溶液的HepG2细胞HK2、BCL2L1 mRNA相对表达量均降低(Р均<0.05)。与空白对照比较,加入不同浓度Roc-A培养HepG2细胞HK2、BCL2L1蛋白相对表量均降低(P均<0.05)。与癌组织HK2高表达的肝癌患者比较,HK2低表达的肝癌患者的生存时间更长(Р<0.05)。结论Roc-A促进HepG2细胞的凋亡,200 nmol/L的Roc-A促进HepG2细胞凋亡能力最佳。Roc-A促HepG2细胞凋亡的机制可能为Roc-A抑制HepG2细胞HK2、BCL2L1 mRNA及蛋白的表达。Objective To observe the apoptosis of human hepatoma cell line HepG2 cultured with rocaglamide(Roc-A) and to explore its possible mechanism.Methods ①The apoptosis of HepG2 cells cultured with different concentrations of Roc-A was observed:HepG2 cells in the logarithmic growth phase were divided into five groups and were added with Roc-A solution of 0(blank control),25,50,100 and 200 nmol/L,respectively.Cells in each group were cultured for 24 h,and apoptosis was observed by YF488-Annexin V/PI fluorescence double staining.②The hexokinase Ⅱ(HK2) and anti-apoptotic factor analogue(BCL2 L1) mRNA in HepG2 cells cultured with different concentrations of RocA were detected:HepG2 cells in the logarithmic growth phase were divided into two groups and were added with 0 and 100 nmol/L Roc-A solution,respectively.HK2 and BCL2 L1 mRNA were detected by RT-qPCR after 24 hours of culture.③HK2 and BCL2 L1 proteins were detected in HepG2 cells cultured with different concentrations of Roc-A:HepG2 cells were divided into three groups which were then added with 0,100 and 200 nmol/L Roc-A,respectively.HK2 and BCL2 L1 proteins of HepG2 cells were detected by proteomics mass spectrometry after 24-hour culture.HepG2 cells in the logarithmic growth phase were divided into five groups and were added with Roc-A solution of 0,25,50,100 and 200 nmol/L,respectively.HK2 and BCL2 L1 proteins were detected by Western blotting at 24 hours of culture.④ Analysis of the relationship between HK2 expression in cancer tissues and survival time of patients with liver cancer:Kaplan Meier Plotter cancer database was used to analyze the relationship between HK2 expression and survival time of liver cancer patients.Results The apoptosis rates of HepG2 cells added with 0,25,50,100 and 200 nmol/L Roc-A solution were0.83%±0.21%,9.46%±2.34%,24.68%±4.22%,39.60%±2.16%,and 58.63%±1.62%,respectively,with statistically significant difference(F=8807,P<0.05).In this experiment,the apoptosis rate increased with the increased concentrations of Roc-A solut

关 键 词：楝酰胺 已糖激酶Ⅱ 抑凋亡因子类似物 细胞凋亡 肝肿瘤 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]