【作者】 熊红；

【导师】 林茂芳；

【作者基本信息】 浙江大学 ， 内科学， 2003， 博士

【摘要】 众所周知，骨髓增生异常综合征（Myelodysplastic syndrome，MDS）难治，尤其是高危MDS。原因可能与MDS恶性克隆对药物作用的敏感性降低有关。目前，MDS的治疗目的是消除或减少瘤细胞克隆，部分或完全重建正常造血，或诱导异常细胞向正常细胞分化成熟。为研究MDS细胞对药物治疗的反应，我们引进了对各种常用的分化诱导剂均无反应的MDS-RAEB型细胞株MUTZ-1。已有研究表明，以拓扑异构酶Ⅱ为作用靶点的第二代蒽环类抗肿瘤药物——阿克拉霉素（aclacinomycin，ACM）治疗某些MDS患者有效。体外实验研究表明，与其它的抗肿瘤剂相比，更不容易产生耐药性，其机理尚不明确。本文旨在研究ACM对MDS-RAEB型细胞株MUTZ-1细胞的体外治疗效应及其新的作用机理。试图证明ACM可诱导MUTZ-1细胞凋亡及（或）向成熟分化，以减少MDS恶性克隆；并试图阐明ACM抗MDS细胞的又一分子机制，从而为克服MDS细胞对化疗药物的耐受寻找新的治疗靶点，为设计新的治疗策略拓宽思路。 采用MTT比色法检测结果显示，0.05μmol/L ACM处理48h，即明显抑制MUTZ-1细胞的增殖，与空白对照组相比，差别有统计学意义（p＜0.05）。ACM作用48h的半数抑制浓度(IC₅₀)为0.46μmol/L。为探讨这种抑制作用的机制，我们从细胞凋亡及细胞分化两个层面作了进一步的研究。 在体外，本研究采用瑞特-姬姆萨染色，光学显微镜观察ACM作用前后MUTZ-1细胞形态，结果显示，0.5μmol/L ACM处理后，MUTZ-1细胞出现凋亡细胞典型的形态学特征， 浙江大学博士学位论文如胞浆空泡化，细胞体积缩小，核染色质固缩，核边聚，染色质断裂，出芽，凋亡小体形成等（图I－2）。 为了对细胞凋亡现象进行准确地定性或定量分析，我们采用多种经典、特异而敏感的实验指标，并利用近年来发展起来的流式细胞分析技术研究了细胞DNA含量、亚二倍体及磷脂酞丝氨酸（PS）转位。结果显示，0．25Pmol／L－0．SPmol／L ACM作用 48h，可见典型的 DNA梯形条带：采用 DNA片断原位末端（TLINEL法）标记方法证明，在一定的作用时间点，随ACM浓度的增加，TUNEL阳性细胞也增加；一定的药物浓度范围内，随ACM作用时间延长，TUNEL阳性细胞增加。从生物化学角度进一步证明，在体外，ACM能诱导MUTZ—l细胞凋亡，且呈剂量一，时间一效应依赖关系。 采用流式细胞仪分析技术探讨了ACM作用前后MUTZ－1细胞DNA含量及亚二倍体的改变。结果显示，0．in mol／L l．2卜mol／L ACM处理后，MUTZ－1细胞周期 G。／M期细胞增多，GllG。期细胞减少，细胞被阻滞于GZ／M期。同时亚二倍体细胞增多，MUTZ－l细胞亚GI期峰更明显(图1．小表明，ACM可能通过阻滞MUTZ－l细胞于细胞周期的GZ／M期，最终导致MUTZ－l细胞凋亡。进一步采用AnnexinV－PI双标记法流式细胞仪分析，结果显示，o．印(、！几＊＊M作用36h，早期凋亡细胞百分数(2！．83叨高于空白对照组（3．n叨；晚期凋亡细胞在处理组仅稍高于空白对照组（厂9％VS 2．65％）。表明，一定浓度 ACM作用一定时间，主要是诱导MUTZI细胞发生早期凋亡（图1．8）。 为研究ACM诱导MUTZ－！细胞凋亡的可能机制及信号传导途径，本研究采用转染报告基因载体pNF。B－SEAP后化学发光测定法分析ACM作用前、后核转录因子NF。B活性水平，并用 RT－PCR法在转录水平检测其调控的靶基因 clAPI及 clAPZ mRNA表达的改变；WCstern blot法检测 ACM作用前、后 NF。B抑制蛋白质 I。B a水平表达的改变，并探讨了ACM较少产生耐药性的可能原因。 研究结果显示，一定浓度 ACM（0．5 u mol／L）处理 MUTZ－l细胞不同时间（3h、6h、12h、24h），导致了抗凋亡基因 clAP－l、cIAPZ mRNA表达水平以时间依赖的方式降低，并与MUTZ－l细胞TUNEL阳性细胞百分数呈负相关（rs－0．991，－0．975）；不同浓度ACM（0三卜mol／L十0 u mol／L）处理 24h，可引起 MUTZ－l细胞 cIAPI、cIAPZ mRNA表达以浓度依赖的方式下调，并与其TUNEL阳性细胞百分数呈负相关（，－0．984，－0．951）。说明ACM诱导MUTZ－l细胞凋亡与抗凋亡基因 clAPI、cIAPZ mRNA表达下调有关。 3 浙江大学博土学位论文 迢过瞬时转染报告基因载体pNF。B－SEAP后化学发光法检测培养上清液中报告基因表达产物 SEAP活性，其活性代表相应转录因于 NF。B活性。结果显示，0．sapOUL ACM作用 3h，MUTZ一！细胞 SEAP活性（8．72土 2．88）较同一时间点空白对照组（1184士 3．11）的为低（cd．2 12，Pq）刀42）；0．SPmol／L ACM作用 6h时，MUTZ—l细胞 SEAP活性与在相应时间点的空白对照间的差异非常显著（P＜0刀1），0．5卜m＊儿＊＊M作用至24卜时，细胞％AP活性己降至相应空白对照组的二．9％。因此，0．5卜mol／L ACM作用 MUTZ—l细胞 3－24h，以时间依赖方式抑制报告基因SEAP活性，当0门卜mol／L ACM处理24h，MUTZ—l细胞SEAP活性(11．84士2．67?更多还原

【Abstract】 It’s well known that patients with MDS, especially with high-risk MDS, have poor prognosis, which may be related to decreased sensibility of MDS malignant clone to drugs. The aim of current therapy of MDS is to reduce, or even erase the malignant clone, reconstruct normal hematopoiesis partly or completely, and induce the abnormal cells differentiation into normal hematopoietic cells. In order to investigate the responses of MDS cells to drugs, a cell line MUTZ-1, derived from a patient with MDS-RAEB. has been introduced to our series, which has no response to traditional differentiation-inducing drugs. It’s reported that aclacinomycin ( ACM ), the second-generation anthracyclines targeting at topoisomerase II, can be used to treat some MDS patients and acquire some response. Compared with other anti-tumor drugs, ACM doesn’t tend to cause drug resistance in experimental study in vitro, but the mechanisms remain unknown. The aim of this paper is to investigate the effect of ACM on MUTZ-1 cell line derived from MDS-RAEB and its new mechanism, try to prove that ACM can induce MUTZ-1 cells afoptosis and (or) differentiation and maturation, so as to reduce the malignant clone of MDS, and try to clarify another new molecular mechanism of ACM on MDS cells. Thereby itwould help to find a new treatment target and develop new strategy to overcome the drug resistance of MDS cells to chemotherapeutic agents.Using MTT assay we investigated the effect of ACM on MUTZ-1 growth. The result showed that following treatment with ACM(0.05 μmol/L) for 48 h, the proliferation of MUTZ-1 cells was inhibited significantly, in comparison with the blank control group(P<0.05). IC50 was 0.46μ mol/L at 48h. To explore the possible mechanism of the inhibition of ACM on MUTZ-1 cells, further investigation was carried out from the two stratifications of inducing apoptosis and differentiation.Using Wright-Giemsa stain, the morphology of MUTZ-1 cells was observed with or without ACM treatment under a light microscope. The result displayed that a series of typical morphological features of apoptosis were observed following treatment with ACM(0.5μmol/L) for 48h, such as vacuolization of cell plasma, shrinkage of cell and nucleus, condensation of nuclear chromatin, marginated against the nuclear membranes, karyorrhexis and convolution ofnuclear, and membrane-bound apoptotic bodies(Figi.2).In order to exactly analyze the cell apoptosis quantitatively and qualitatively, in addition tosome classic and specific and sensitive methods, the flow cytometry was also used to evaluate the DNA content, percentage of hypodiploid and translocation of phosphatidyl serine( PS). It showed that following treatment with 0.25umol/L-0.5nmol/L ACM for 48h, a typical DNA ladder was observed. With TdT mediated-dUTP nick end labeling (TUNEL), it appeared that with the increasing concentration of ACM at a certain time-point, the TUNEL positive cells increased. Meanwhile, with the increasing treatment time at a certain concentration of ACM, the TUNEL positive cells increased. From biochemistry, it was proved that ACM could induce MUTZ-1 cells apoptosis with dose- and time- dependent manners in vitro.With the help of flow cytometry, the DNA content and hypodiploid changes of MUTZ-1 cells were observed with or without ACM treatment. The results showed that" the percentage of MUTZ-1 cells in the G2/M phase of the cell cycle increased, while in the G1/G0 phase decreasedfollowing treatment with 0.1μ mol/L 0.2μmoI/L ACM, indicating that MUTZ-1 cells were arrested in the Ga/M phase. At the same time, hypodiploid cells increased, and the sub-G| peak appeared (Fig1-7). This manifested that ACM at a certain concentration could arrest MUTZ-1 cells in the G2/M phase of cell cycle, and then induced the cells apoptosis. With the help of flow cytometry following annexinV-PI staining, exposure to 0.5nmol/L ACM for 36 h caused 21.83% early apoptotic cells, which was more than that of the untreated group (3.47%), while late apoptovic cells increased only sligh更多还原