【作者】 张婷；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科学， 2013， 博士

【摘要】 研究背景白血病是起源于造血系统的常见恶性肿瘤,严重危害着人类健康。但是,其发病原因和疾病发展的确切病理机制迄今仍未阐明。目前,化学药物联合化疗依然是临床上大多数白血病患者最常用的治疗方法。高三尖杉酯碱是由中国科研人员于20世纪70年代首先从中草药南方粗榧碱中提取出来的抗白血病生物碱,在临床上已经被广泛应用于急性髓系白血病的治疗,并取得了良好的疗效。此外,多项临床试验表明,高三尖杉酯碱在伊马替尼治疗失败的慢性髓系白血病患者中仍然具有治疗价值,尤其是在T315I突变的慢粒治疗中起重要作用,提示该药物可作为慢性髓系白血病患者的挽救治疗选择。目前药物作用的分子机制尚不清楚,现有研究认为,高三尖杉酯碱抗肿瘤活性主要与抑制蛋白合成和诱导凋亡两大机制有关。但是,这些研究都不能回答该药物直接作用的靶蛋白是什么。因此,为了进一步明确该药的具体作用机制,我们在本研究中制备了高三尖杉酯碱亲和柱,通过亲和柱结合白血病细胞蛋白来寻找药物直接结合的靶蛋白；并通过建立靶蛋白高表达的细胞系,采用表达谱芯片技术、实时定量PCR以及免疫印迹等手段与方法来探讨高三尖杉酯碱通过靶蛋白发挥抗白血病作用的可能分子机制。研究方法1. 细胞培养：(1)人白血病细胞培养：髓系白血病细胞系Kasumi-1、单核细胞白血病细胞系THP1和U937、慢性髓系白血病细胞系K562；(2)逆转录病毒包装细胞：经改造后用于逆转录病毒包装的293细胞系Plat-GP细胞。2.细胞增殖活力检测：MTS法3. 细胞周期检测：流式细胞技术4.细胞凋亡检测：流式细胞技术,PI/Annexin-V标记物染色法检测。5. 亲和柱制备：采用链亲和素琼脂糖亲和树脂以及生物素进行制备,经色谱-质谱技术检测。6.蛋白洗脱与鉴定：采用PBS、NaCl及高三尖杉酯碱溶液洗脱,纯化干燥后获得蛋白,经聚丙烯酰胺凝胶电泳后染色,切割并消化蛋白条带后,通过蛋白质谱技术对靶蛋白进行分析鉴定。7.构建稳定转染细胞株：采用逆转录病毒进行转染实验,通过嘌呤霉素筛选稳转株,并经过western blot验证。8.表达谱芯片分析：采用Affymetrix GeneChip(?) Human Genome U133 Plus 2.0 Array芯片检测和分析稳定转染细胞系靶蛋白高表达后的基因表达谱的变化情况。9. 实时定量PCR：检测和验证芯片结果以及观察经高三尖杉酯碱处理后白血病细胞系的RNA表达变化情况。10. Western blot分析：检测和验证芯片结果以及检测经高三尖杉酯碱作用后白血病细胞系的蛋白表达变化情况。结果1.高三尖杉酯碱对急性髓系白血病细胞系THP1、U937、和Kasumi-1细胞以及慢性髓系白血病细胞系K562有明显的生长抑制作用,作用48小时的IC50值分别为22.37 ng/ml、11.09 ng/ml、14.93 ng/ml和37.38 ng/ml。2.高三尖杉酯碱能够诱导人细胞白血病细胞系发生凋亡,药物作用后凋亡细胞百分比明显上升。3.高三尖酯碱亲和柱结合的直接靶蛋白为actin-结合蛋白non muscle myosin heavy chain IIA (NMHC IIA),并且高三尖杉酯碱作用后可上调其在细胞中的表达水平。4.表达谱芯片结果提示,白血病细胞系稳定高表达NMHC IIA蛋白后,可引l起基因表达谱发生改变,主要表现为细胞周期和MAPK激酶通路相关基因表达水平的上调。5.实时定量PCR和Western Blot实验结果与表达谱芯片结果基本符合,主要表现为NMHC IIA蛋白高表达后,细胞周期相关蛋白(主要为G1期→S期转化相关蛋白)的上调,以及高三尖杉酯碱作用后细胞MAPK激酶通路上c-jun和JNK的激活和上调,可能由此促发凋亡程序的启动。结论1.高三尖杉酯碱能够抑制白血病细胞系的生长与增殖,并且促进肿瘤细胞凋亡的发生。2.高三尖杉酯碱作用白血病细胞时,直接与NMHC IIA发生结合并上调其蛋白表达水平3.高三尖杉酯碱通过上调NMHC IIA,影响细胞周期相关蛋白的表达,促进细胞周期向S期转换,从而为高三尖杉酯碱与其它化疗药物的联合提供了理论基础。4.高三尖杉酯碱通过上调NMHC IIA,激活MAPK通路中JNK/c-jun激酶途径,上调JNK凋亡信号传导通路来启动细胞凋亡。更多还原

【Abstract】 BackgroudLeukemia is a common malignant neoplasm of hematopoietic system. However, its pathogenesis and molecular mechanism of development is still not clear. In recent years, combination chemotherapy is one of most frequently means used to treat leukemia in clinical practice.Homoharringtonine (HHT) is a natural alkaloid that extracted from Cephalotaxus fortunei. Its antitumor activity was first reported by Chinese investigators in the 1970s. It has been proved that this agent used to be considered the most effective treatment for acute myelogenous leukemia. In addition, a series of clinical trials confirmed the therapeutic value of HHT for patients with chronic myeloid Leukemia (CML) after failure on imatinib therapy, suggesting homoharringtonine could be used as salvage therapy for patients who have imatinib-resistant CML. So far, little is known about the molecular mechanism of its action. Preclinical studies demonstrate that HHT exerts its antitumor activity via inhibition of protein synthesis and induction of cell apoptosis. However, these studies could not answer the question that what is the direct target protein of HHT.In order to further determine the specific mechanism of the drug, we identified the direct target proteins of HHT by preparing the affinity column in this study. We also established human leukemia cells stably expressing high levels of the target protein and employed a series of technologies, including gene-chip array, real-time PCR and immune, to explore possible mechanisms of HHT in leukemia.Materials and Methods1. Cell culture:(1) Human leukemia cell lines:acute monocytic leukemia cell lines, THP1 and U937; acute myelogenous leukemia cell line, Kasumi-1; chronic myelogenous leukemia cell line, K562;(2) Plat-GP cells:a 293T-derived murine-leukemia-virus-based packaging cell line.2. Cell proliferation assay with MTS assay.3. Cell cycles assay of leukemia cell line with Flow cytometry.4. Apoptosis assay of leukemia cell line with Flow cytometry and PI/Annexin-V assay.5. Prepare the affinity column of HHT with Streptavidin agarose resins and biotin and identify by LC-MS mass.6. Protein elution and identification:wash the affinity column with PBS, NaCl and HHT solutions respectively; dialyze and dry the target proteins; stain the protein bands on SDS-PAGE; excise and digest the protein bands; identify the target proteins through protein Mass spectrum.7. Establishment of cells stably over-expressing the target protein:cells were transduced by infection with retrovirus-containing supernatants of transfected Plat-GP cells and selected with puromycin in maintenance medium.8. Gene expression assay in leukemia cell line stably over-expressing the target protein with Affymetrix GeneChip(?) Human Genome U133 Plus 2.0 Array.9. Assay of mRNA expression in leukemia cells after treatment with HHT by real-time PCR.10. Protein expression assay in leukemia cells after treatment with HHT by Western blot.Results1. Homoharringtonine inhibited significantly the proliferation of acute myelogenous leukemia cell lines, THP1, U937 and Kasumi-1, and chronic myelogenous leukemia cell line, K562. The values of IC50 were 22.37 ng/ml,11.09 ng/ml,14.93 ng/ml and 37.38 ng/ml at 48 hours respectively.2. Homoharringtonine induced cell apoptosis. The percentage of apoptosis cells after treatment with HHT increased obviously.3. Actin-binding protein, Non muscle myosin heavy chain IIA, is the direct target protein of HHT by preparing HHT affinity column. HHT also could up-regulate the protein.4. Results of microarray showed that over-expressing NMHC IIA could influence expression levels of signal molecules in cell cycle and MAPK pathways in leukemia cells.5. Western Blot and RT-PCR confirmed the results of microarray. Either treatment with HHT or high expression of NMHC IIA could up-regulate the expression level of proteins in cell-cycle and MAPK pathways, such as c-jun and JNK.Conclusion1. Homoharringtonine could inhibit the proliferation of leukemia cell lines and induce apoptosis of these cells.2. Homoharringtonine directly binds and up-regulates the actin-binding protein, non muscle myosin heavy chain IIA.3. Homoharringtonine affects expression levels of cell cycle-related proteins via up-regulation of NMHC IIA, promoting Gl→S cell phage transiton.4. Activation and up-regulation of JNK/c-jun signaling pathway via up-regulating NMHC IIA might be one of mechanisms for Homoharringtonine-induced apoptosis.更多还原