单独及联合转染survivin和bcl-2反义寡核苷酸诱导胆囊癌细胞凋亡的研究  

Inhibition of survivin and bcl-2 Antisense Oligodeoxynucleotides Combined Transfection on the Human Gallbladder Carcinoma Cell Line GBC-SD in vitro

作　　者：冯立民[1] 王建立[1] 乌新林[1] 姜希宏[1] 寿楠海[1] 

机构地区：[1]山东大学齐鲁医院普外科,济南250012

出　　处：《中国肿瘤生物治疗杂志》2004年第4期248-251,共4页Chinese Journal of Cancer Biotherapy

摘　　要：目的:探讨单独及联合转染survivin、bcl-2反义寡核苷酸(AsODN)对胆囊癌细胞系GBC-SD的凋亡促进作用。方 法:设计并合成特异性靶向survivin及bcl-2的AsODN。免疫组织化学法观察胆囊癌细胞中Survivin及Bcl-2蛋白的表达情 况;实验分为4组:空白对照组、survivin AsODN转染组、bcl-2 AsODN转染组、联合survivin及bcl-2 AsODN转染组。转染24 h 后,采用逆转录-聚合酶链反应(RT-PCR)检测survivin基因表达的变化,电镜下观察细胞形态变化,流式细胞仪技术检测细胞 凋亡率,四甲基偶氮唑蓝(MTF)法检测AsODN对细胞的生长抑制率。结果:Survivin及Bcl-2蛋白在胆囊癌细胞中均有较高 表达;单独及联合转染survivin及bcl-2 AsODN后,胆囊癌细胞内survivin基因mRNA表达下调47.8％;,电镜下胆囊癌细胞呈 典型凋亡样改变;凋亡率分别为11.38％±3.91％(survivin AsODN组)、9.26％±4.15％(bcl-2 AsODN组)、28.45％±6.34％ (联合转染组);分别与对照组相比差异有显著性(P<0.01),而联合转染组同单独转染survivin AsODN组及bcl-2 AsODN组相 比差异也有显著性(P<0.05)。各转染组相对于空白对照组的AsODN抑制率分别为54.3％,47.6％,76.5％。结论:Survivin 及Bcl-2蛋白在胆囊癌细胞中呈高表达。Objective: To investigate the effect of survivin and bcl-2 antisense oligodeoxynucleotides ( AsODN) combined transfection on the human gallbladder carcinoma cell line GBC-SD in vitro. Methods: Survivin and Bcl-2 protein expressions were detected by immunohistochemical method; Cultured cells were divided into 4 groups: Nomal control group, survivin antisense observed group, bcl-2 antisense observed group and combined group. After transfected for 24 h, the expression of survivin mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Cell morphological changes were observed under electron microscopy. Apoptosis index (AI) was examined by flow cytometry; Inhibitory rate (IR) was determined by the colorimetri MTT cell viability and proliferation assay. Results: Survivin and Bcl-2 protein were highly expressed in gall bladder carcinoma cells; The expression of survivin mRNA was decreased 47. 8%. Abnormal morphological changes of cells were observed in the three AsODN transfection groups; The AI in survivin antisense observed group,bcl-2 antisense observed group,and combined group was 11.38%±3.91% , 9.26%±4.15% , 28.45%±6.34% respectively and significantly higher than the nomal control group (P<0. 01) , and that in the combined group was significantly higher than the other two observed groups(P <0.05). The IR was 54.3% ,47.6% ,76.5% respectively in the three AsODN transfection groups. Conclusion: Survivin and bcl-2 protein were highly expressed in gallbladder carcinoma cells. Survivin AsODN and bcl-2 AsODN can induce GBC-SD cell apoptosis and the effect was more markedly by combining the two AsODNs.

关 键 词：胆囊癌 SURVIVIN BCL-2 寡核苷酸类 反义 转染 

分 类 号：R735.8[医药卫生—肿瘤]