【作者】 蔡真；

【导师】 林茂芳；

【作者基本信息】 浙江大学 ， 内科学（血液病）， 2001， 博士

【摘要】 [目的]高三尖杉酯碱（Homoharringtonine，HHT）是从我国特有的植物海南粗榧分离的有效抗癌药物。我国在世界上首先用于急性非淋巴细胞白血病（ANLL）的临床治疗，疗效显著，HHT对慢性髓细胞性白血病同样有效。近年，国内外对HHT的作用机制的研究发现HHT有抑制蛋白合成、克隆形成以及诱导细胞分化的作用；对~3H标记的胸腺嘧啶核苷掺入DNA也有影响。研究还发现HHT能促K562细胞c-myc基因上调，启动凋亡；在凋亡后期能使c-fos基因表达增加，表明HHT促细胞凋亡是受基因调控的。HHT在体外能诱导HL-60、K562和Jurkat细胞的细胞凋亡，但未进一步阐述其促凋亡的机理。迄今，人们对HHT促凋亡作用限于髓系白血病的研究，而HHT作用于T淋巴细胞白血病的促凋亡及其机理研究尚未见报道。 通过对HHT致白血病细胞凋亡的研究，将有助于深入地了解化疗药物对肿瘤细胞凋亡及其信号传导途径的调控，为进一步提高HHT的临床疗效，扩大HHT的临床应用范围提供理论依据和研究思路。[方法］1 应用流式细胞仪技术、末端脱氧核糖核酸片段标记和细胞染色方法，研究HHT 浙江大学博士学位论文促人 T－淋巴细胞白血病细胞株 Molt3（P53“）、MO卜16（P53”）、Ichikawa（P53”’）、Jurkat（P53”’）和 HL－60（P53－）细胞凋亡、细胞周期的改变及其与抑癌基因 P53的关系。2 流式细胞仪检测细胞APO表面抗原表达及抗FAS和抗FAS配体作用后的凋亡、检测线粒体膜电位的改变、ROS产生和抗氧化剂的作用；比色法检测caspase－3活性及caspase－3－8、－9抑制剂影响HHT对线粒体膜电位改变和细胞凋亡。3 半定量逆转录酶－多聚酶链式反应检测 HHT对细胞内 Bax和 Bcl－2 mRNA表达的调控。4 通过共聚焦激光扫描显微镜分析，线粒体示踪剂 Mito Tracker、Bax和细胞色素C单克隆抗体及与带有荧光cy－5的羊抗鼠u抗作用，研究Bax和细胞色素C在HHT作用前后的细胞内的定位。［结果］IHHT能诱导T－白血病细胞凋亡，具时间和剂量依赖性，且与P53存在形式相关，影响细胞周期，阻滞细胞在 GI期 HHT（Zn旮l、10nyml、25 nyml、50 n旮ml）分别作用于 Mort3（P53“）、Moltl6（P53“）；Ichikawa（P53”’）、Jurkat（P53”’）细胞和 HL－60（P53－）24小时，Molt3、Moltl6细胞凋亡率随着剂量加大凋亡率增加；Ichikawa和 Jurkat细胞在 HHT相同剂量时的凋亡率比 Molt3、Moltl 6f氏午值分别＜0刀5）；而H卜60细胞经HH T作用后，其凋亡率明显低于M。1G、讪1在16 o值分别＜O刀5），与 Ichikawa和 Jurkat的凋亡率无明显差异厂值分别＞0刀5）。将不同浓度 HHT分别作用于 Molt3细胞 6、12、18、24小时，其凋亡率随着时间延长而增加。2 HH”H不影响FAS受体／配体（FAS－R／FAS－L）对Molt3细胞的凋亡诱导作用HHT作用MOlt－3细胞后的相对荧光密度显示，HHT无诱导FASK表达增强作用；细胞经抗人FAS单抗CHll作用后，显示凋亡，但HHT对CHll无协同或增强作用；FAS配体中和抗体（NOKI、2）对 HHT作用的细胞凋亡无任何阻断作用。 2一3 HHT作用后细胞线粒体膜电位（么wm）的改变 荧光染料 JC＊显示线粒体膜电位的存在和下降。当 MOltS细胞经 HHT 25n咖1处理12h后，线粒体膜电位下降，荧光发散由 3％增加至 42％，与细胞凋亡的百分率相近，推测 HHT诱导细胞凋亡是与线粒体膜电位下降有关。4 HHT作用后细胞内细胞色素 C定位的改变在未经 HHT处理的细胞内，细胞色素 C位于线粒体内，HHT 25ng／il处理细胞 12h后则弥散地分布在 Annexin－V阳性的早期凋亡细胞的胞浆内。5 HHT通过激活 caspasel诱导细胞凋亡 在 HHT10ng／ml和 50pgipl水平，可观察到Motr3细胞的caspase〕活性随着剂量加大而增强，同时，casPase〕活性也随着 HHT作用时间延长而随之增加。用 caspase上、8、－9特异性抑制齐 Z－DEVD－FKK。Z．IETD－FMK ＄［IZ．LEHD．FMK先于 HHT与细胞作用，发现 CSSp＆SC－3、－8、－9抑制剂均能不同程度地抑制 Molt3细胞的凋亡；经caspasel抑制剂处理后，能部分抑制 HHT处理后的 Ichikawa细胞凋亡，而 caspase｛、刁抑制剂无抑制作用；结果还发现么mm几乎不受caspase上、七、习抑制剂影响。说明八pm在casPases作用的上游；但caspase－8、－9抑＄剂言抑伟casnase3的活性，表明casPase－3在casPase－8、－9的下游。6 HHT处理的细胞有活性氧（ROS）产生 抗氧化剂 NAC预处理细胞，可观察到NAC能减少ROS的产生。然而，抗氧化剂并不影响HHT对线粒体膜电位及CCPCSS3活性，也不抑制HHT诱导的细胞凋亡；7 Bcl－2、BAX mRNA表达与 HHT的作用的关系 HHT能上调 Molt3细胞BAX表达、下调 Bc1E表达；IChik洲。细胞 BAX、BCI工的表达在 HHT作用前后未发生 改变：8 B盼在HHT诱导细胞凋亡过程的表达和细胞内定位 经HHT 25ng／ffil作用12h后，BSX即易位到线粒体内。细胞更多还原

【Abstract】 OmECTrvEHomoharringtonine (HHT) is a cephalotaxine alkaloid isolated from genusCephalotaxus. HHT was first introduced to clinic for treatment of leukemia inl970’ in China and has been proven to have strong ami-leukemia activity It isused as an effective anti-leukemia drug clinica1ly in treatment of acute myeloidleukemia as well as chronic myeloid leukemia. In the last few decades, studieshave shown that the inhibitory effects of HHT on leukemia cell groWth includeinhibition of protein synthesis, decrease of clonogenicity and induction ofleukemia cell differentiation. As evidenced by ’H-Thymidine incopofationassnys, HHT also demonstrated the capacity to inhibit DNA synthesis inleukemia cells. Recent studies indicated that HHT is caPable of uP-regulating ofc-myc and c-fos, consequently promoting aPoptosis in myeloid leukemia cellssuch as K562 cells. Although the HHT’s effect on inducing aPoptosis in cellline models such as HL-60, K562 and Jurkat is obvious, the detailed mechanismof HHT in regulation of aPoptosis in leukemia is not yet clear To date, little isknOwn aboul the role that HHT plays in regUlation of aPoptosis in lymphocyticleukemia. The study on the mechanisms of HHT in regulation of aPoptosis inleukemia cells will give us insigh into further understanding of the role HHTmay play in regulation of aPoptosis in leukemia cel1s and to underiine thesignaling pNay that HHT may interact with duing aPoPt()sis induced by thechemotheraPelltic drug. The data generated from this Stlldy will also providevaluable information for designing chemotheraPeutical regimens in clinicaltreatment of lymphocytic leukemia and to improve the clinical olltcome ofpatients receiving anii-leukemic chemotheraPyMATERIALS AND METHODS1 In this study, four T-cell leukemia cell 1ines Mo1t-3, Molt~l6,Ichikawa,Jurkat and one myeloid cell line HL-60 were used in the experimentsto detect the effects of HHT on induction of aPoptosis and to study the possiblemechanisms of HHT in regulation of aPoptosis of the cells. Wth the excePtionof Molt-3 and Moltl6 cells, which expressed a wild type of p53, the other twolines expressed mutant-type p53 in Ichikawa and Jurkat, null p53 expressed inHL-60. Cell cycle was analyzed by fluorescence-activated Flow Cytometry(FACS); DNA breakage resulting from HHT treatment was detected by TdTtransferase labeling. The morphologic observation using Giemsa staining wasmade on various cells treated by HHT.2 FACS analysis was also used fOr detection of FAS protein expressed oncell surface, for evaluation of aPoptosis in the cells after treatment with ani-Fasand anti-Fas ligand (Fas-L ) aniibodies, for detCction of mitochondrialmembrane potential (MMP ) and the generation of reactive oxygen species(ROS ) in the cells, as well as the effect of ami-oxidants on genefation of ROSin the cells tfCated with HHT Caspase-3 activity as well as the effects of theinhibitors of CasPase-3, 8, 9 on MMP and on aPoPtosis in the cells induced byHHT was also evaluated.3 Semi-quanitative RT-PCR was used to evaluate the mRNA expression ofBax and Bcl-2 in the cells treated with HHT4 By labelling the cells with Mito Tracker Red CM-H,XROS and stainingsubsequently with ani-cytochrome C and anti-Bax anibody the subcellularlocalization and re-distribution of Bax and cytochrome C in cells after treatmentWth HHT was analyzed by Confocal Laser Scan MicroscopyussUrrsl The results demonstfated that HHT was caPable of inducing aPoptosis in theT-cell leukemia cells in time- and dose-dependam manners. The statlls of p53has an effect on cell cycles of the cells as Gl arrest was observed in the two T-cell lines with wild-type p53 and Ichikawa cells with mutant p53 aftertreatmellt with HHT Twniy-four hours after tfCattheflt of Molt-3, Molt-l6,Ichikawa, Jurkat and HL-60 cells with HHT at different doses of 2ng/ml,10ng/ml, 25ng/ml and 50ng/ml, significan aPoptotic cells were detected in theMol更多还原