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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 decrease of Bcl - 2 Bax protein ratio is a checkpoint for the initiation of apoptosis induced by radiation. In this study, Bax antisense nucleic acid was used to interfere with the expression of Bax gene, increase the ratio of Bcl 2 Bax protein, inhibit radiation-induced apoptosis and promote cell survival. METHODS: HELF cells and mouse bone marrow cells were treated with Bax antisense oligodeoxynucleotides immediately after irradiated by γ-rays in human embryonic lung fibroblasts (HELF) and mice for 6h (final concentration was 100nmol L) MTT assay, SRB assay and mouse bone marrow neutrophil-macrophage cell line (CFU-GM) colony formation assay to determine the cell survival. The expression of Bax mRNA and cell cycle were detected by RT-PCR and flow cytometry, and the mechanism of action was analyzed. Results: The survival rate of HELF cells increased by 24.4% (MTT assay, P <0.01) and 12.8% (SRB assay, P <0.01), respectively. The number of CFU-GM colony formation in bone marrow cells (72 ± 17 vs 13 ± 6, P <0.01). The results of RT-PCR showed that the ratio of Bax β_actin in the target band and the internal standard band of the treatment group was lower than that of the control group 4 3.9% and 6 8.6% respectively). Flow cytometry showed that the proportion of cells in the total S phase in the treated group was higher than that in the control group by 24.2% and 7.7%, respectively, suggesting that the number of Bax mRNA copies in the treated group was low In the control group, cell proliferation activity is strong. CONCLUSIONS: Antisense thio-deoxynucleotides targeting Bax mRNA can promote the survival of cells after γ-ray irradiation, and their mechanism may destroy the target mRNA and then decrease the expression of Bax protein, inhibit the initiation of apoptosis and promote cell proliferation related.