Bax反义硫代脱氧寡核苷酸的体外抗辐射效应研究  被引量:1

In vitro anti-radiation effects of antisense thiodeoxyoligonucleotides targeted to Bax mRNA

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作  者:曹智刚[1] 袁守军[1] 陈惠鹏[1] 徐兰平[1] 田增月[1] 韩昌明[1] 丁林茂[1] 

机构地区:[1]军事医学科学院放射医学研究所,北京100850

出  处:《军事医学科学院院刊》2004年第1期11-15,共5页Bulletin of the Academy of Military Medical Sciences

基  金:全军"十五"指令课题 ( 0 1L0 2 2 )

摘  要:目的 :Bcl_2 Bax蛋白比值降低是辐射所致细胞凋亡启动的检查点。本研究拟用Bax反义核酸干预Bax基因的表达 ,提高Bcl_2 Bax蛋白比值 ,抑制辐射所致凋亡 ,促进细胞存活。方法 :人胚肺成纤维细胞 (HELF)和小鼠受γ射线照射后 ,立即用Bax反义脱氧寡核苷酸处理HELF细胞和小鼠骨髓细胞 6h(终浓度均为 10 0nmol L) ,用MTT法、SRB法和小鼠骨髓中性粒细胞_巨噬细胞系祖细胞 (CFU_GM)集落形成法 ,确定细胞的存活状况。用RT_PCR方法和流式细胞仪检测BaxmRNA的表达状况、细胞周期的变化 ,分析其作用机制。结果 :HELF细胞的存活率分别增加 2 4 .4 % (MTT法 ,P <0 .0 1)和 12 .8% (SRB法 ,P <0 .0 1) ;骨髓细胞处理组CFU_GM集落形成数量远高于照射对照(72± 17vs 13± 6 ,P <0 .0 1) ;RT_PCR检测显示 ,处理组的目标条带和内标条带的灰度比值 (Bax β_actin)低于对照组(分别为 4 3.9%和 6 8.6 % ) ;流式细胞仪检测表明 ,处理组总S期细胞的比例高于对照组 ,分别为 2 4 .2 %和 7.7% ,提示处理组细胞内BaxmRNA拷贝数量低于对照组 ,细胞增殖活动较强。结论 :靶向BaxmRNA的反义硫代脱氧寡核苷酸 ,能够促进γ射线辐射后细胞的存活 ,其机制与破坏靶mRNA分子 ,继而造成Bax蛋白表达减少 ,抑制凋亡启动并促进细胞增殖活动有关。Objective:The ratio of Bcl-2/Bax proteins is a key checkpoint of apoptotic initiation by radiation. The goal of the present study was to increase the ratio of Bcl-2/Bax proteins through reducing translation of Bax protein by antisense thiodeoxyoligonucleotides targeted to Bax mRNA, and thus to inhibit radiation-induced apoptosis and to enhance cell survival.Methods:Immediately after exposure of human embryonic lung fibroblast (HELF) and mice to γ-radiation, the HELF and mouse bone marrow cells were treated with Bax antisense deoxyoligonucleotides for 6 h (final concentration was 100 nmol/L). The survival profile of HELF was determined with MTT and SRB assay, the mouse CFU-GM with CFU-GM colony formation. With RT-PCR and flow cytometry, the alteration of Bax mRNA expression and cell cycle of HELF were examined in order to explore its mechanisms of action.Results:The survival rate of treated HELF increased by 24.4% (MTT) and 12.8% (SRB) (P<0.01). The count of CFU-GM colonies in treated group was significantly higher than that in control (72±17 vs 13±6, P<0.01). The result of RT-PCR showed that the ratio between Bax and β-actin in treated group and control was 43.9% and 68.6%, respectively. The proportion of total S-phase of HELF of treated group and control was 24.2% and 7.7% determined by flow cytometry. The above suggested that the number of copies of Bax mRNA be lower in treated cells than in control cells, and the treated cells may have more active proliferation. Conclusions: The antisense thiodeoxyoligonucleotedes could promote the survival of cells exposed to γ-ray, and its mechanisms might involve reducing expression of Bax protein secondary to damage of target mRNA, inhibiting apoptotic initiation and enhancing active proliferation.

关 键 词:辐射损伤 细胞凋亡 BAX 反义 硫代脱氧寡核苷酸 抗辐射 细胞存活 

分 类 号:R96[医药卫生—药理学]

 

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