摘要
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 explore the possible mechanisms of cell apoptosis induced by arsenic trioxide （ATO） alone or in combination with thalidomide （THAL）on SCID mice model transplanted with human myelodysplastic syndrome （MDS） cell line MUTZ-1 in vivo. Methods In a previous study by the authors, a Hum-MDS mice model was successfully established and the results showed that ATO alone or in combination with THAL had markedly inhibitory effect on the tumors growth in the MDS mice model of the treated groups with higher-rates of cell apoptosis and longer-survival periods than control groups （P 〈 0.01 ）. In the present study, 48 Hum-MDS mice were randomly grouped and ATO groups had 32 mice which were treated with ATO [5.0/μg or7. 5 /μg/（g·d） , 5 d a week, intraperitoneal injection （i. p. ） ×3 weeks alone or in combination with THAL 8 μg/（g · d） × 3 weeks] and the control groups had 16 mice [treated with 0. 9% NaC1 10 μl/（g·d）（ i. p） or untreated ]. The expressions of the proteins and the genes related to apoptosis were detected by using immunohistochemical （IHC） method, Western blot, reverse transcription-polymerase chain reaction （ RT-PCR ） , electrophoretic mobility shift assay （ EMSA ） with [ γ-32P ] ATP oligonucleotide probe end labeling. Results （ 1 ） By IHC, compared to controls, the expressions of the proapoptosis protein Bax （ F = 1080. 150, P 〈 0. 01 ） and the ratio of Bax/Bcl-2 were higher, but Bcl-2 （F =2777. 978,P 〈0. 01）was lower in the treated groups. （2） Western-blot results showed that the proapoptosis protein expressions of mitochondrial Smac/DIABLO and cytochrome C （IHC） were strikingly upregulated and pro-caspase 9, 7, 6, 3 and full PARP were down-regulated and cleaved caspase-6,7,9 and PARP were clearly observed in the treated groups. The expressions of apoptosis protein inhibitors of cIAP1 and survivin mRNA （RT-PCR） were markedly down-regulated in the treated groups. But no evidences of cleavage of BID and caspase-8 had been observed in any group. （ 3 ） The expression of IκB-α protein [ nuclear factor -kappa B（NF-κB） inhibitor] was up-regulated（Western-blot） and the activity of NF-κB were sharply down-regulated in ATO-treated groups by EMSA. Conclusions These results suggest that mitochondria are the primary intracellular target of ATO. At least, two pathways, namely, mitochondrial mediated-caspase dependent cell apoptosis pathway and NF-κB signal conduct pathway were involved in the mechanisms of ATO inducing cell apoptosis on MDS mice model in vivo. The mechanisms seem to be a complicated network regulated by multi-signal pathways, multi-layers, and multi-factors instead of a single pathway.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2006年第10期782-786,共5页
Chinese Journal of Pediatrics
基金
浙江省科学技术厅重点资助项目(2003C23013)
