摘要
目的:比较胡桃醌对人乳头瘤病毒(HPV)阳性宫颈癌Caski细胞、HPV阴性C33A细胞和敲减脯氨酰顺反异构酶1(Pin1)基因的Caski细胞(shCaski细胞)增殖的抑制作用,探讨其可能的作用机制。方法:构建表达Pin1 shRNA慢病毒载体用于感染细胞,筛选得到稳定的shCaski细胞。取对数生长期的Caski、shCaski和C33A细胞,分为正常对照组、10、20、50和100μmol·L^(-1)胡桃醌组,胡桃醌处理24 h后,采用MTT法检测各组的细胞增殖活性。取对数生长期的Caski细胞和C33A细胞,分为对照组和20μmol·L^(-1)胡桃醌组,各组细胞处理24 h后,流式细胞术检测各组不同周期细胞百分率和早期凋亡率,Hoechst33258荧光染色观察各组细胞形态表现,Western blotting法检测各组细胞周期和凋亡相关蛋白表达水平。结果:MTT实验,与正常对照组比较,不同浓度胡桃醌组Caski细胞增殖活性明显降低(P<0.05或P<0.01);50和100μmol·L^(-1)胡桃醌组C33A和shCaski细胞增殖活性明显降低(P<0.05)。Hoechst 33258染色,与对照组比较,20μmol·L^(-1)胡桃醌组Caski细胞和C33A细胞中均可观察到核碎解增加,Caski细胞核碎解数量较C33A细胞多。流式细胞术检测,与对照组比较,20μmol·L^(-1)胡桃醌组Caski细胞G2期细胞百分率明显升高(P<0.01),20μmol·L^(-1)胡桃醌组C33A细胞G2期细胞百分率差异无统计学意义(P>0.05);与对照组比较,20μmol·L^(-1)胡桃醌组Caski细胞早期凋亡率明显升高(P<0.01),20μmol·L^(-1)胡桃醌组C33A细胞早期凋亡率差异无统计学意义(P>0.05);Western blotting法检测,与C33A细胞比较,Caski细胞中Pin1蛋白表达量明显升高;胡桃醌组Caski细胞和shCaski细胞中Pin1蛋白表达量明显低于对照组;与对照组比较,胡桃醌组C33A细胞中细胞周期相关蛋白表达水平明显改变;Caski细胞中磷酸化ATM(Patm)、磷酸化细胞周期检查点激酶2(pChk2)、216位丝氨酸磷酸化细胞分裂周期蛋白25c(pCdc25c Ser216)和pCdc25c Tyr216蛋白表�
Objective:To compare the inhibitory effects of juglone on the proliferation of human papilloma virus(HPV)-positive cervical cancer Caski cells,negative C33A cells and Caski cells knocked down the Pin1 gene(shCaski cells),and to explore its possible mechanism.Methods:A lentiviral vector expressing Pin1 shRNA was constructed and used to infect the cells,and shCaski cells were obtained.The Caski,shCaski and C33A cells in the logarithmic growth phase were selected and divided into normal control group and 10,20,50 and 100μmol·L^(-1)juglone groups.After juglone treatment for 24 h,the cell proliferation activities in various groups were detected by MTT method.The Caski cells and C33A cells in the logarithmic growth phase were selected and divided into control group and 20μmol·L^(-1)juglone group.After 24 h of treatment of the cells in each group,the percentages of cells in different cell cycles and the early apoptotic rates in various groups were detected by flow cytometry.Hoechst33258 fluorescence staining was used to observe the morphology of nucleus;Western blotting method was used to detect the expressions of cell cycle and apoptosis-related proteins.Results:The MTT experiment showed that the proliferation activities of the Caski cells in different concentrations of juglone groups were significantly decreased(P<0.05 or P<0.01)compared with normal control group,while the proliferation activities of C33A and shCaski cells in 50 and 100μmol·L^(-1)juglone groups were significantly decreased(P<0.05).The results of Hoechst 33258 staining showed that compared with control group,both the Caski cells and C33A cells in 20μmol·L^(-1)juglone group showed increased nuclear fragmentation,and the number of nuclei fragmentation of Caski cells was more than that of C33A cells.The results of flow cytometry showed that compared with control group,the percentage of Caski cells in G2phase in 20μmol·L^(-1)juglone group was increased significantly(P<0.01);while there was no significant difference in the percentage of C33A cells
作者
赵行宇
杨欣
朱志华
何涵
宋梓桐
张巍
ZHAO Xingyu;YANG Xin;ZHU Zhihua;HE Han;SONG Zitong;ZHANG Wei(Department of Biochemistry,Jilin Medical College,Jilin 13201,China;Department of Biochemistry,School of Basic Medical Sciences,Yanbian University,Ynabian 133000,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2022年第2期348-355,共8页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科技发展计划项目(20190701062GH)
吉林省教育厅“十三五”科学技术研究项目(JJKH20200453KJ)。
