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目的探讨三氧化二砷(arsenictrioxide,ATO)单用或联合沙利度胺(Thalidomide,THAL)体内诱导人骨髓增生异常综合征(myelodysplasticsyndromes,MDS)荷瘤小鼠细胞凋亡的可能途径和机制。方法用人MDS荷瘤小鼠模型作为实验动物,将48只荷瘤小鼠随机分为治疗组32只(ATO单用或联合THAL)和对照组16只。采用免疫组织化学(immunohistochemistry,HIC)、Western-blot、逆转录-聚合酶链反应(RT-PCR)、电泳迁移率实验(electrophoreticmobilityshiftassay,EMSA)等多参数方法,检测各组荷瘤组织凋亡相关蛋白和基因的表达水平。结果治疗后2周,(1)IHC检测显示,与对照组比较,治疗组Bcl-2家族促凋亡蛋白Bax表达水平上调(F=1080.150,P<0.01);而抗凋亡蛋白Bcl-2表达水平下调(F=2777.978,P<0.01),Bax/Bcl-2比值上升。提示ATO诱导细胞凋亡与Bcl-2家族凋亡相关蛋白表达及表达比有关。(2)Western-blot发现,治疗组线粒体促凋亡蛋白Smac/DIABLO和细胞色素C(IHC)的表达比对照组明显上调;半胱天冬酶(caspase)9、7、6、3及其下游底物聚ADP核糖聚合酶(PARP)蛋白全长被剪切,表达明显下调,可见caspase9、7、6和PARP激活带表达。提示ATO能通过线粒体介导caspase依赖的途径诱导荷瘤细胞凋亡。(3)治疗组凋亡蛋白抑制因子cIAP蛋白(Western-blot)和SurvivinmRNA(RT-PCR)的表达比对照组明显下调。(4)EMSA实验显示,与对照组比较,治疗组核因子kappaB(NF-kB)活性表达显著下调;而IkB-α(NF-kB抑制子)蛋白表达明显上调(Western-blot).提示ATO诱导荷瘤细胞凋亡涉及NF-kB信号途径。结论ATO体内诱导人MDS荷瘤小鼠细胞凋亡至少通过两条重要的途径:一是线粒体介导-Caspase依赖途径;二是NF-kB信号途径,这两条途径相对独立,又在一定水平上相互关联。认为ATO体内诱导细胞凋亡过程复杂,是多条途径、多个水平、多个步骤和多种因子之间相互协调的网络效应,并非单一途径。实验为临床治疗MDS提供了新的思路。
Objective To investigate the possible pathways and mechanism of apoptosis induced by arsenictrioxide (ATO) alone or in combination with thalidomide (THAL) in murine models of myelodysplastic syndromes (MDS). Methods Using human MDS tumor-bearing mouse model as experimental animal, 48 tumor-bearing mice were randomly divided into treatment group (ATO alone or in combination with THAL) and control group (16). The apoptosis of tumor-bearing tissues in each group was detected by multi-parameter methods such as immunohistochemistry (HIC), Western-blot, reverse transcription-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assay (EMSA) Protein and gene expression levels. Results 2 weeks after treatment, (1) IHC showed that compared with the control group, the Bcl-2 family pro-apoptotic protein Bax was up-regulated (F = 1080.150, P <0.01) (F = 2777.978, P <0.01), and the ratio of Bax / Bcl-2 increased. Tip ATO induced apoptosis and Bcl-2 family apoptosis related protein expression and expression ratio. (2) The expression of mitochondrial pro-apoptotic proteins Smac / DIABLO and cytochrome C (IHC) in the treatment group was significantly higher than that in the control group by Western-blot. The expressions of caspase 9, 7, 6, The downstream substrate poly ADP ribose polymerase (PARP) protein was cut the full length, the expression was significantly down, showing caspase 9,7,6 and PARP activation band expression. Tip ATO can induce apoptosis of tumor-bearing cells through mitochondria-mediated caspase-dependent pathway. (3) Western blot and Survivin mRNA (RT-PCR) expression in the treatment group were significantly lower than the control group. (4) The results of EMSA showed that the expression of nuclear factor kappaB (NF-kB) in the treatment group was significantly down-regulated compared with the control group, whereas the expression of IkB-α (NF-kB inhibitor) Induction of apoptosis in tumor-bearing cells involves the NF-kB signaling pathway. Conclusion ATO induced apoptosis in human MDS tumor-bearing mice through at least two important pathways: one is mitochondrial-Casspase-dependent pathway; the other is NF-κB signaling pathway, which is relatively independent and at a certain level On each other. ATO is considered to be a complex pathway that induces apoptosis in many ways. It is not the only way to co-ordinate network effects at multiple levels, multiple steps and multiple factors. The experiment provides a new idea for the clinical treatment of MDS.