摘要
目的研究第二线粒体源的半胱天冬酶激活物(Smac)基因联合顺铂对人肝癌细胞SMMC-7721中半胱氨酸天冬氨酸蛋白酶(caspase)-3、caspase-9和细胞色素c(Cyt c)蛋白表达的影响。方法利用脂质体介导的方法将Smac转染入SMMC-7721,G418筛选后采用逆转录-聚合酶链式反应(RT-PCR)和蛋白印迹(western blot)法检测筛选所得SMMC-7721/Smac细胞中Smac mRNA和蛋白的表达,四甲基偶氮噻唑蓝(MTT)法绘制细胞生长曲线;顺铂处理后采用免疫细胞化学法检测细胞中caspase-3、caspase-9和Cyt c蛋白的表达。结果 SMMC-7721/Smac细胞中Smac mRNA和蛋白表达均明显增加,12～120 h A490值为0.28～0.70,均明显低于同一时间点的SMMC-7721(0.31～1.10)(P<0.05);与SMMC-7721比较,SMMC-7721/Smac细胞中caspase-3和Cyt c蛋白表达均增高,累积光密度值(IOD)分别从19 939/14 924增至28 347/21 017(P<0.05);顺铂作用下,SMMC-7721和SMMC-7721/Smac细胞中caspase-3、caspase-9和Cyt c蛋白表达均增加,且后者更明显;剂量为25μg/mL时,SMMC-7721/Smac的caspase-3、caspase-9的IOD分别从对照的28 347/22 412增至46 696/39 728(P<0.001)。结论 Smac稳定过表达联合顺铂可明显增强细胞中凋亡相关蛋白caspase-3、caspase-9和Cyt c表达,从而促进细胞凋亡。
Objective To study effects of the cotreatment of second mitochondria-derived activator caspase（Smac）and cisplatin on the expressions of caspase-3,caspase-9,and cytochrome c（Cyt c）in hepatocellular carcinoma cells（SMMC-7721）.Methods SMMC-7721 cells were transfected with Smac by lipofectamine-mediated method and then screened by G418.Smac mRNA and protein expressions in SMMC-7721/Smac cells acquired by G418 screen were determined by reverse transcription-PCR（RT-PCR）and western blot and its growth curve was performed by methyl thiazolyl tetrazolium（MTT）assay.After cisplatin treatment,expressions of caspase-3,caspase-9,and Cyt c were evaluated by western blot.Results Expressions of both Smac mRNA and its protein in SMMC-7721/Smac cells increased significantly,and its A490 value was significantly lower than that of SMMC-7721 cells in the same time-point（P0.05,P0.001）.Protein expression detection showed that expressions of caspase-3 and Cyt c increased in SMMC-7721/Smac cells compared with that of SMMC-7721 cells,from 19 939 to 28 347 and 14 924 to 21 017,respectively（P0.05-P0.001,with F values of 12.588 and 23.811）.Cisplatin treatment increased expressions of caspase-3,caspase-9 and Cyt c in both SMMC-7721 and SMMC-7721/Smac cells,especially in Smac cells.After the treatment of 25 μg/ml cisplatin,expressions of caspase-3 and caspase-9 increased from 28 347 to 46 696 and 22 412 to 39 728,respectively（P0.001,F with values of 12.588 and 12.180）.Conclusion Cotreatment of stable Smac over-expression and cisplatin could increase cellular expressions of caspase-3,caspase-9,and Cyt c,which could promote cell apoptosis.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2012年第10期1315-1317,共3页
Chinese Journal of Public Health
基金
国家自然科学基金青年基金(30700672)
