穿山甲蛋白提取物对人白血病K562细胞增殖与凋亡的影响
|
摘要
【研究背景】
慢性粒细胞白血病(Chronic myeloid leukemia, CML)是血液系统常见的恶性肿瘤之一。化疗仍然是治疗白血病的主要手段,其副作用及临床耐药已成为化疗的主要障碍。酪氨酸激酶抑制剂(Tyrosine kinase inhibitors, TKI)的出现是内科治疗CML的新的里程碑,但是由于基因变异引起的耐药现象也日见增多,因此寻找针对CML更有效、毒副反应更少的药物是临床治疗迫切需要解决的问题之一。
传统中药治疗肿瘤由来已久。现代中医学认为肿瘤的病机多为“热毒”、“瘀血”、“瘀毒”等,从近几十年来所取得的成果来看,砷剂(雄黄)、苦参碱(苦参)、靛玉红(青黛)等均作为“以毒攻毒”、“清热解毒”、“活血化瘀”的药物而被应用于中医临床,并取得显著疗效。目前,用现代化的科学技术,对传统中草药进行大力开发,对其药理学作用给予新的理解和阐述,致力于研究开发出新的抗癌药物,来大力弘扬祖国传统医学,已成为当前中医药研究的发展趋势。
现代研究证实中药治疗白血病的作用可表现在以下几方面：直接诱导细胞凋亡,一定程度逆转白血病多药耐药,增加患者免疫力,减轻化疗副作用等。白血病与凋亡关系密切,利用凋亡机制清除白血病细胞引发出白血病治疗的新观念、新手段。中药可通过多种途径诱导白血病细胞凋亡,包括直接杀伤、细胞周期阻滞、影响凋亡基因表达、诱导分化、提高细胞因子及体内激素水平等。
现代药理研究表明穿山甲对肿瘤的影响包括一定程度抑制肿瘤生长,使T细胞亚群、NK细胞活性增高,提高晚期癌症患者生存质量及免疫功能,减少患者恶心呕吐、白细胞减少、血小板减少等化疗副作用。临床研究用于治疗肺癌、乳腺癌、白血病等恶性肿瘤均有报道。但临床使用穿山甲时,多与其他中药配伍使用,单用或提取成分应用较少,而且对慢性粒细胞白血病的作用及作用机制的报道目前仍未见。本研究进一步验证穿山甲蛋白提取物的体外抗白血病疗效,为穿山甲的临床使用奠定实验基础。
【研究目的】
为进一步验证穿山甲对白血病的抗肿瘤作用,以慢性髓细胞性白血病K562细胞株为研究对象,观察穿山甲蛋白提取物对K562细胞的生长增殖及凋亡的影响,探讨穿山甲诱导K562细胞凋亡可能的作用机制。
【研究方法】
1.采用四甲基偶氮唑盐(MTT)法检测K562细胞增殖。
2. Hoechst 33258荧光染色法观察K562细胞凋亡的形态变化。
3.流式细胞仪检测K562细胞的凋亡率,验证穿山甲蛋白提取物诱导K562细胞凋亡的作用。
【研究结果】
1.MTT实验观察穿山甲蛋白提取物对K562细胞生长及增殖的影响
各浓度穿山甲蛋白提取物对K562细胞的增殖有不同程度的抑制作用,与空白对照组比较有显著性差异(P<0.01),且在一定剂量范围内,随穿山甲蛋白提取物浓度的增加,其抑制率越来越高,具有明显的剂量依赖性；24小时IC50是12.07ug／ml；提取液本身有一定促凋亡作用,但与一定浓度穿山甲蛋白提取物相比,差异有统计学意义(P<0.05)。
2. Hoechst 33258荧光染色法观察K562细胞的形态变化
20μg／ml剂量的穿山甲蛋白提取物作用K562细胞48 h后在镜下可见细胞皱缩、变形,染色质凝聚且边缘化,部分细胞体积变小并呈现DNA荧光碎片或出现浓染致密的块状和颗粒状强荧光。
3.流式细胞术分析细胞凋亡
Annexin V-PI双染色法检测结果显示未加药组、单纯提取液组、穿山甲蛋白提取物组48小时凋亡率分别是(3.42±0.69)%、(35.32±1.45)%、(49.04±1.84)%,穿山甲蛋白提取物组凋亡率高于阴性对照组和单纯提取液组(P<0.05)。穿山甲蛋白提取物组与单纯提取液组的坏死率无显著差异。
【结论】
1.不同浓度穿山甲蛋白提取物能够不同程度抑制K562细胞增殖,并且抑制率呈明显的剂量-效应关系。
2. Hoechst 33258荧光染色法显示穿山甲蛋白提取物能够诱导细胞凋亡,呈特征性的细胞凋亡形态。
3. Annexin-PI双染色法检测凋亡率进一步证实了穿山甲蛋白提取物诱导K562细胞凋亡的作用。
[Background]
Chronic myeloid leukemia (CML) is one of the common malignant tumors in the blood system. Chemotherapy remains the primary treatment of CML, but its side effects and clinical drug resistance have become major obstacles to chemotherapy. The emergence of Tyrosine kinas inhibitors (TKI) is a new milestone in the treating of CML, but there is an increasing number of drug resistance because of genetic mutations, so looking for more effective and lower toxic drugs against CML in clinical treatment is an urgent need to address this problem.
The treatment of cancer using traditional Chinese medicine has a long history. Modern Chinese medicine believes that the pathogenesis of most tumors are" heat-toxicity ", " stasis", "stasis-toxicity ", etc..The results has suggested in recent decades that arsenic (realgar), matrine (Sophora) and indirubin (indigo naturalis) are thus as a " using poison to counteract poison "," clearing away heat and toxic material", " Promoting blood circulation and removing blood stasis " drugs to be applied to clinical medicine, and have had remarkable clinical efficacy. Currently, with modern science and technology, develop strongly on traditional Chinese herbal medicine, understand and elaborate the pharmacology, research and develop new anti-cancer drugs, and carry forward the Chinese traditional medicine vigorously, have become the medical research trends.
Modern studies confirmed the role of the treatment of traditional Chinese medicine of leukemia which can be shown in the following areas:direct induction of apoptosis, to some extent to reverse the multi-drug resistance of leukemia, increase the immunity of patients and reduce side effects of chemotherapy. Leukemia is closely related to apoptosis of leukemia cells and using apoptosis mechanism to clear leukemia cells rise new concepts, new methods. Chinese medicine can induce apoptosis of leukemia cells in a variety of ways, including kill leukemia cells directly,arrest cell cycle, influence apoptosis gene expression, induce differentiation and increase levels of cytokines and other hormones.
Modern pharmacological studies show that the pangolin has impact on tumor growth including a certain degree of inhibition of tumor, increasing activity of T cell subsets and NK cell, improving the quality of patients'life and immune function, reducing nausea and vomiting, leukopenia, thrombocytopenia and other side effects of chemotherapy. Clinical researches about the treatment of lung cancer, breast cancer, leukemia and other malignancies have been reported. Pangolin has usually applied with other medicine, used alone or its extract is seldom found, and its effect of chronic myeloid leukemia and the mechanism are still can not be seen. This study wants to validate pangolin protein extract has effect of anti-leukemia in vitro, and lays the foundation of clinical use of pangolin.
[Objective]
To further verify the anti-leukemia effect of pangolin, using chronic myeloid leukemia K562 cell lines as study object, to observe the effect of pangolin protein extract on the proliferation and apoptosis of K562 cells, to explore the mechanism of pangolin which induces apoptosis of K562 cells.
[Methods]
1. MTT assay was used to observe the effects on growth inhibition induced by pangolin protein extract in K562 cells.
2. The morphological changes of K562 cells induced by pangolin protein extract were observed with Hoechst 33258 staining and transmission electron microscope.
3. The percentage of apoptotic cells was detected by flow cytometry, in order to verify the effect of pangolin protein extract on apoptosis.
[Results]
1. MTT experimental observation of pangolin protein extract on K562 cell growth and proliferation
All kinds of concentrations of pangolin protein extract could inhibit K562 cell proliferation strongly, compared with the control group there were significant differences (P<0.01), and in a certain dose range, with increasing concentration of pangolin protein extract, The inhibition rates were higher,appeared dose-dependent; 24-hour IC50 is 12.07ug/ml; The extract itself had some apoptosis ability, but compared the pangolin protein extract, the difference was statistically significant (P <0.05).
2. Hoechst 33258 staining was used to observe the morphological changes of K562 cells
20μg/ml dose of pangolin protein extract effected on K562 cells for 48 h,in the microscope,cell shrinkage,deformation,chromatin condensation and marginalization, some cells became smaller and showed DNA fragmentation or appeared fluorescent stain dense block and strong granular fluorescence.
3. Apoptosis was analyzed by flow cytometry
Annexin V-PI double staining showed that the 48-hours apoptosis rates of the negative control group,the extract control group and pangolin protein extract group were (3.42±0.69)%, (35.32±1.45)%, (49.04±1.84)%, the apoptosis rate of pangolin protein extract group higher than the negative control group and the extract control group (P<0.05). Pangolin protein extract group and the pure extract group showed no difference in necrosis.
[Conclusions]
1. Pangolin protein extract could inhibit the K562 cells on some degrees, and inhibition rates appeared dose-dependent.
2. Hoechst 33258 staining assay showed that pangolin protein extract could induce apoptosis, showed characteristic morphology of apoptosis.
3. Annexin V-PI double staining further confirmed pangolin protein extract can induce apoptosis of K562 cells.
慢性粒细胞白血病(Chronic myeloid leukemia, CML)是血液系统常见的恶性肿瘤之一。化疗仍然是治疗白血病的主要手段,其副作用及临床耐药已成为化疗的主要障碍。酪氨酸激酶抑制剂(Tyrosine kinase inhibitors, TKI)的出现是内科治疗CML的新的里程碑,但是由于基因变异引起的耐药现象也日见增多,因此寻找针对CML更有效、毒副反应更少的药物是临床治疗迫切需要解决的问题之一。
传统中药治疗肿瘤由来已久。现代中医学认为肿瘤的病机多为“热毒”、“瘀血”、“瘀毒”等,从近几十年来所取得的成果来看,砷剂(雄黄)、苦参碱(苦参)、靛玉红(青黛)等均作为“以毒攻毒”、“清热解毒”、“活血化瘀”的药物而被应用于中医临床,并取得显著疗效。目前,用现代化的科学技术,对传统中草药进行大力开发,对其药理学作用给予新的理解和阐述,致力于研究开发出新的抗癌药物,来大力弘扬祖国传统医学,已成为当前中医药研究的发展趋势。
现代研究证实中药治疗白血病的作用可表现在以下几方面：直接诱导细胞凋亡,一定程度逆转白血病多药耐药,增加患者免疫力,减轻化疗副作用等。白血病与凋亡关系密切,利用凋亡机制清除白血病细胞引发出白血病治疗的新观念、新手段。中药可通过多种途径诱导白血病细胞凋亡,包括直接杀伤、细胞周期阻滞、影响凋亡基因表达、诱导分化、提高细胞因子及体内激素水平等。
现代药理研究表明穿山甲对肿瘤的影响包括一定程度抑制肿瘤生长,使T细胞亚群、NK细胞活性增高,提高晚期癌症患者生存质量及免疫功能,减少患者恶心呕吐、白细胞减少、血小板减少等化疗副作用。临床研究用于治疗肺癌、乳腺癌、白血病等恶性肿瘤均有报道。但临床使用穿山甲时,多与其他中药配伍使用,单用或提取成分应用较少,而且对慢性粒细胞白血病的作用及作用机制的报道目前仍未见。本研究进一步验证穿山甲蛋白提取物的体外抗白血病疗效,为穿山甲的临床使用奠定实验基础。
【研究目的】
为进一步验证穿山甲对白血病的抗肿瘤作用,以慢性髓细胞性白血病K562细胞株为研究对象,观察穿山甲蛋白提取物对K562细胞的生长增殖及凋亡的影响,探讨穿山甲诱导K562细胞凋亡可能的作用机制。
【研究方法】
1.采用四甲基偶氮唑盐(MTT)法检测K562细胞增殖。
2. Hoechst 33258荧光染色法观察K562细胞凋亡的形态变化。
3.流式细胞仪检测K562细胞的凋亡率,验证穿山甲蛋白提取物诱导K562细胞凋亡的作用。
【研究结果】
1.MTT实验观察穿山甲蛋白提取物对K562细胞生长及增殖的影响
各浓度穿山甲蛋白提取物对K562细胞的增殖有不同程度的抑制作用,与空白对照组比较有显著性差异(P<0.01),且在一定剂量范围内,随穿山甲蛋白提取物浓度的增加,其抑制率越来越高,具有明显的剂量依赖性；24小时IC50是12.07ug／ml；提取液本身有一定促凋亡作用,但与一定浓度穿山甲蛋白提取物相比,差异有统计学意义(P<0.05)。
2. Hoechst 33258荧光染色法观察K562细胞的形态变化
20μg／ml剂量的穿山甲蛋白提取物作用K562细胞48 h后在镜下可见细胞皱缩、变形,染色质凝聚且边缘化,部分细胞体积变小并呈现DNA荧光碎片或出现浓染致密的块状和颗粒状强荧光。
3.流式细胞术分析细胞凋亡
Annexin V-PI双染色法检测结果显示未加药组、单纯提取液组、穿山甲蛋白提取物组48小时凋亡率分别是(3.42±0.69)%、(35.32±1.45)%、(49.04±1.84)%,穿山甲蛋白提取物组凋亡率高于阴性对照组和单纯提取液组(P<0.05)。穿山甲蛋白提取物组与单纯提取液组的坏死率无显著差异。
【结论】
1.不同浓度穿山甲蛋白提取物能够不同程度抑制K562细胞增殖,并且抑制率呈明显的剂量-效应关系。
2. Hoechst 33258荧光染色法显示穿山甲蛋白提取物能够诱导细胞凋亡,呈特征性的细胞凋亡形态。
3. Annexin-PI双染色法检测凋亡率进一步证实了穿山甲蛋白提取物诱导K562细胞凋亡的作用。
[Background]
Chronic myeloid leukemia (CML) is one of the common malignant tumors in the blood system. Chemotherapy remains the primary treatment of CML, but its side effects and clinical drug resistance have become major obstacles to chemotherapy. The emergence of Tyrosine kinas inhibitors (TKI) is a new milestone in the treating of CML, but there is an increasing number of drug resistance because of genetic mutations, so looking for more effective and lower toxic drugs against CML in clinical treatment is an urgent need to address this problem.
The treatment of cancer using traditional Chinese medicine has a long history. Modern Chinese medicine believes that the pathogenesis of most tumors are" heat-toxicity ", " stasis", "stasis-toxicity ", etc..The results has suggested in recent decades that arsenic (realgar), matrine (Sophora) and indirubin (indigo naturalis) are thus as a " using poison to counteract poison "," clearing away heat and toxic material", " Promoting blood circulation and removing blood stasis " drugs to be applied to clinical medicine, and have had remarkable clinical efficacy. Currently, with modern science and technology, develop strongly on traditional Chinese herbal medicine, understand and elaborate the pharmacology, research and develop new anti-cancer drugs, and carry forward the Chinese traditional medicine vigorously, have become the medical research trends.
Modern studies confirmed the role of the treatment of traditional Chinese medicine of leukemia which can be shown in the following areas:direct induction of apoptosis, to some extent to reverse the multi-drug resistance of leukemia, increase the immunity of patients and reduce side effects of chemotherapy. Leukemia is closely related to apoptosis of leukemia cells and using apoptosis mechanism to clear leukemia cells rise new concepts, new methods. Chinese medicine can induce apoptosis of leukemia cells in a variety of ways, including kill leukemia cells directly,arrest cell cycle, influence apoptosis gene expression, induce differentiation and increase levels of cytokines and other hormones.
Modern pharmacological studies show that the pangolin has impact on tumor growth including a certain degree of inhibition of tumor, increasing activity of T cell subsets and NK cell, improving the quality of patients'life and immune function, reducing nausea and vomiting, leukopenia, thrombocytopenia and other side effects of chemotherapy. Clinical researches about the treatment of lung cancer, breast cancer, leukemia and other malignancies have been reported. Pangolin has usually applied with other medicine, used alone or its extract is seldom found, and its effect of chronic myeloid leukemia and the mechanism are still can not be seen. This study wants to validate pangolin protein extract has effect of anti-leukemia in vitro, and lays the foundation of clinical use of pangolin.
[Objective]
To further verify the anti-leukemia effect of pangolin, using chronic myeloid leukemia K562 cell lines as study object, to observe the effect of pangolin protein extract on the proliferation and apoptosis of K562 cells, to explore the mechanism of pangolin which induces apoptosis of K562 cells.
[Methods]
1. MTT assay was used to observe the effects on growth inhibition induced by pangolin protein extract in K562 cells.
2. The morphological changes of K562 cells induced by pangolin protein extract were observed with Hoechst 33258 staining and transmission electron microscope.
3. The percentage of apoptotic cells was detected by flow cytometry, in order to verify the effect of pangolin protein extract on apoptosis.
[Results]
1. MTT experimental observation of pangolin protein extract on K562 cell growth and proliferation
All kinds of concentrations of pangolin protein extract could inhibit K562 cell proliferation strongly, compared with the control group there were significant differences (P<0.01), and in a certain dose range, with increasing concentration of pangolin protein extract, The inhibition rates were higher,appeared dose-dependent; 24-hour IC50 is 12.07ug/ml; The extract itself had some apoptosis ability, but compared the pangolin protein extract, the difference was statistically significant (P <0.05).
2. Hoechst 33258 staining was used to observe the morphological changes of K562 cells
20μg/ml dose of pangolin protein extract effected on K562 cells for 48 h,in the microscope,cell shrinkage,deformation,chromatin condensation and marginalization, some cells became smaller and showed DNA fragmentation or appeared fluorescent stain dense block and strong granular fluorescence.
3. Apoptosis was analyzed by flow cytometry
Annexin V-PI double staining showed that the 48-hours apoptosis rates of the negative control group,the extract control group and pangolin protein extract group were (3.42±0.69)%, (35.32±1.45)%, (49.04±1.84)%, the apoptosis rate of pangolin protein extract group higher than the negative control group and the extract control group (P<0.05). Pangolin protein extract group and the pure extract group showed no difference in necrosis.
[Conclusions]
1. Pangolin protein extract could inhibit the K562 cells on some degrees, and inhibition rates appeared dose-dependent.
2. Hoechst 33258 staining assay showed that pangolin protein extract could induce apoptosis, showed characteristic morphology of apoptosis.
3. Annexin V-PI double staining further confirmed pangolin protein extract can induce apoptosis of K562 cells.
引文
[1]Ren R.Mechanisms of BCR-ABL in the pathogeniesis of chronic myelogenous leukemia [J].Nature Reviews Cancer,2005,5 (3):172-183.
[2]程志.从砷剂治疗白血病获效探索抗肿瘤中药的研究方向[J].浙江中医杂志,2005,3：98-99.
[3]Wyllic, A.H, Kerr, J.F.R, Curric, AR. Cell death:the significance of apoptosis[J]. Int Rev Cytol,1980,68:251-256.
[4]Lieberthal W, Levine J.S. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury[J]. Am J Physiol,1996,271:F477-12.
[5]Richard S. H, Andreas S, Jonathan E. M, et al. Mechanisms of disease cell death[J]. N Engl J Med,2009,361:1570-1583.
[6]谢新生,张秀丽,赵家军等.穿山甲煎液诱导HL 260细胞凋亡的研究[J].浙江中西医结合杂志,2001,11(8)：477-479.
[7]郭利华,李 艺.李斯文教授运用穿山甲治疗血液病的经验[J].云南中医中药杂志,2009,30(11)：1-2.
[8]张东伟,付 敏,彭贤文等.猪蹄甲与穿山甲抗小鼠骨髓微循环障碍作用的实验研究.中草药,2005,36(9)：1364-1367.
[9]许重远,李亦蕾,陈振德等.高效毛细管蛋白电泳法对鸡内金和穿山甲的鉴别[J].解放军药学学报,2007,23(6)：464.
[10]Kwong YL,Todd D.Dclicious poison:arscnic trioxide for the treatment of leukemia[J].Blood,1997,89(9):3487.
[11]Zhang P. The use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia[J]. J Biol Regul Homeost Agents,1999,13(4):195-200.
[12]Thomas X, Troncy J. Arsenic:a beneficial therapeutic poison-a historical overview[J]. Adler Mus Bull.2009 Jun;35(1):3-13.
[13]Wang H.Combined effect of docetaxel and cisplatin for non-small cell lung cancer cell lines in vitro[J].Nagoya Med Sci,2000,63(3-4):129-137.
[14]陈志炉,史亦谦,周郁鸿.中药诱导白血病细胞凋亡的研究近况[J].实用中西医结 合临床2007,7(3)：91-92.
[15]陈婷梅,祝彼得.抗白血病中药及天然药物的研究现状[J].中国中西医结合杂志,1995,15(5)：317-320.
[16]Zhang XW, Yan XJ, Zhou ZR, et al. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML[J].Science.2010,328(5975): 240-243.
[17]Cheng XD, Hou CH, Zhang XJ, et al. Effects of Huangqi (Hex) on inducing cell differentiation and cell death in K562 and HEL cells[J].Acta Biochim Biophys Sin (Shanghai).2004 Mar;36(3):211-217.
[18]于淼,李娜,胡丽娜等.动物药整理研究——穿山甲[J].吉林中医药,2009,29(6)：514-516.
[19]盂庆常.穿山甲治疗白细胞减少症应用体会[J].甘肃中医,2009,22(8)：27.
[20]Guo ZM, Li HJ, Qian XH. Gamma-globin synthesis in K562 cells induced with Tortois plastron, Astragali, Salviae miltiorrhizae and Codonopsis pilosulae[J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi,2008 Jun;16(3):520-524.
[1]Ren R.Mechanisms of BCR-ABL in the pathogeniesis of chronic myelogenous leukemia [J].Nature Reviews Cancer,2005,5 (3):172-183.
[2]高莉,李晓明.酪氨酸激酶抑制剂治疗慢性粒细胞白血病的研究进展[J].中国药房,2008,19(11)：865-867.
[3]Gora-Tybor J, Robak T.Targeted drugs in chronic myeloid leukemia [J].Curr Med Chem,2008,15 (29):3036-3051.
[4]Hehlmann R, Saussele S.Treatment of chronic myeloid leukemia in blast crisis Haematologica[J].2008,93(12):1765-1769.
[5]牛家华,王春.伊马替尼耐药慢性粒细胞白血病治疗进展[J].医药专论-世界临床药物,2008,29(5)：285-288.
[6]刘 鹤,陈晓光.慢性粒细胞白血病治疗中克服伊马替尼耐药的新策略[J].中华肿瘤杂志,2006,28(8)：561-563.
[7]邓雪蓉,王文生.慢性粒细胞白血病对STI571耐药机制的研究进展[J].白血病·淋巴瘤,2005,14(6)：382-384.
[8]PABLO RAMIREZ, JOHN F. DIPERSIO. Therapy Options in Imatinib Failures[J]. The Oncologist,2008,13:424-434.
[9]David Frame. New strategies in controlling drug resistance in chronic myeloid leukemia[J]. Am J Health-Syst Pharm,2007,64 (15):16-21.
[10]Weisberg E, Manley PW, Cowan-Jacob SW, et al. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia[J]. Nat.Rev.Cancer,2007,7:345-356.
[11]Jabbour E, Soverini S. Understanding the role of mutations in therapeutic decision making for chronic myeloid leukemia[J]. Semin Hematol,2009,46(2 Suppl 3):S22-6.
[12]Gisella Volpe, Cristina Panuzzo,Stefano Ulisciani, et al. Imatinib resistance in CML[J].Cancer Letters,2009,274:1-9.
[13]Hochhaus A,Kantarjian HM, Baccarani M, et al.Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy [J].Blood,2007,109:2303-2309.
[14]Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia:an up-date of concepts and management rec-ommendations of European Leukemia Net[J].JClin Oncol.2009,27(35):6041-6051.
[15]H.M. Kantarjian, F. Giles, N. Gattermann, et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and Intolerance [J]. Blood,2007,110:3540-3546.
[16]Michael Steinberg, Pharm D, BCOP. Dasatinib:A Tyrosine Kinase Inhibitor for the Treatment of Chronic Myelogenous Leukemia and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia[J]. Clin Ther,2007,11(29):2289-2308.
[17]ELIAS JABBOUR, JORGE E. CORTES, HAGOP M. KANTARJIAN, Suboptimal Response to or Failure of Imatinib Treatment for Chronic Myeloid Leukemia:What Is the Optimal Strategy?[J].Mayo Clin Proc,2009,84(2):161-169.
[18]Elias Jabbour, Jorge Cortes,Hagop Kantarjian.Treatment selection after imatinib resistance in chronic myeloid leukemia[J].Targ Oncol,2009,4:3-10.
[19]Michael Brave,Vicki Goodman,Edvardas Kaminskas, et al. Sprycel for Chronic Myeloid Leukemia and Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Resistant to or Intolerant of Imatinib Mesylate[J]. Clin Cancer Res,2008,14(2):352-359.
[20]Shah NP, Kantarjian HM, Kim DW, et al. Intermittent target inhibition with dasatinib 100mg once daily preserves efficacy and improves tolerability in imatinib-resistant and intolerant chronic-phase chronic myeloid leukemia[J]. JClinOncol,2008,26(19):3204-3212.
[21]O'Hare T, Walters DK, Stoffregen EP, et al. In vitro activity of BCR-ABL inhibitors AMN107 and BMS-354825 against clinically relevant imatinib resistant Ab1 kinase domain mutants[J].Cancer Res,2005,65:4500-4505.
[22]Michael W. Deininger.nilotinib[J].Clin Cancer Res,2008,4(13):4027-4031.
[23]Kantarjian HM, Giles F, Gatterman N. Nilotinib (formerlyAMN107), a highly selective BCR-ABL tyrosine kinase inhibitor,is effective in patients with Philadelphia chromosome-positivechronic myelogenous leukemia in chronic phase following matinib resistance and intolerance [J]. Blood,2007,110:3540-3546.
[24]Giles FJ, Larson RA, Kantarjian HM, et al. Nilotinib in patients with Philadelphia hromosome-positive chronic myelogenous leukemia in blast crisis who are resistant or intolerant to imatinib(abstract 1025) [J]. Blood,2007,110:310a.
[25]le Coutre P, Ottmann OG, Giles F, et al.Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is active in patients with imatinib-resistant or intolerant accelerated-phase chronic myelogenous leukemia[J]. Blood, 2008,111:1834-1839.
[26]Hughes T, Saglio G, Martinelli G, et al. Responses and disease progression in CML-CP patients treated with nilotinib after imatinib failure appear to be affected by the BCR-ABL mutation status and types [abstract 320] [J]. Blood,2007, 110(11):101a.
[27]DavidL.DeRemer,PharmD,Celalettin Ustun,Kavita Natarajan. Nilotinib:A Second-Generation Tyrosine Kinase Inhibitor for the Treatment of Chronic Myelogenous.Leukemia[J].Clinical Theapeutics,2008,30(11):1956-1975.
[28]Jabbour E, Hochhaus A, Cortes J, et al. Choosing the best treatment strategy for chronic myeloid leukemia patients resistant to imatinib:weighing the efficacy and safety of individual drugs with BCR-ABL mutations and patient history [J].Leukemia.2010,24(1):6-12.
[29]Soverini S, Colarossi S, Gnani A, et al. Resistance to dasatinib in Philadelphia-positive leukemia patients and the presence or the selection of mutations at residues 315 and 317 in the BCR-ABL kinase domain[J].Haematologica.2007,92 (3):401-404.
[30]Shah NP, Skaggs BJ, Branford S, et al.Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency[J].J Clin Invest.2007,117(9):2562-2569.
[31]Khorashad JS, Milojkovic D, Mehta P, et al. In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib[J]. Blood, 2008,111(4):2378-2381.
[32]Shah NP, Kantarjian HM, Kim DW, et al. Intermittent target inhibition with dasatinib 100 mg once daily preserves efficacy and improves tolerability in imatinib-resistant and-intolerant chronic-phase chronic myeloid leukemia[J]. J Clin Oncol.2008,26(19):3204-3212.
[33]antarjian HM, Giles F, Gattermann N, et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance[J].Blood,2007,110(10):3540-3546.
[34]Hochhaus A, Muller MC, Cortes JE, et al. Dasatinib efficacy after imatinib failure by dosing schedule and baseline BCR-ABL mutation status in patients with chronic myeloid leukemia in chronic phase (CML-CP) [J].Haematologica 2008,93: 371-372.
[35]Hughes T, Saglio G, Branford S, et al.Impact of baseline BCR-ABL mutations on response to nilotinib in chronic myeloid leukemia patients in chronic phase (CML-CP) [J] J Clin Oncol,2009,27:4204-4210.
[36]S.Soverini, A. Gnani, S. Colarossi, et al.Philadelphia chromosome-positive leukemia patients who harbor imatinib-resistant mutations have a higher likelihood of developing additional mutations associated with resistance to novel tyrosine kinase inhibitors [J]. Blood,2007,110(11):101a.
[37]Philipple Coutre, Michaela Schwarz, Theo D. Kim, New Developments in Tyro- sine Kinase Inhibitor Therapy for Newly Diagnosed Chronic Myeloid Leukemia [J].Clin Cancer Res,2010,16(6):1771-1780.
[38]Natalia L. Komarova, Allen A. Katouli, Dominik Wodarz.Combination of Two but Not Three Current Targeted Drugs Can Improve Therapy of Chronic Myeloid Leukemia[J].PLoS ONE,2009,4(2):e4423(1-5).
[39]J.Cortes,T. Bruemmendorf, H. Kantarjian,et al.Efficacy and safety of bosutinib (SKI-606) among patients with chronic phase Ph+chronic myelogenous leukemia (CML) [J]. Blood,2007,110(11):311a-312a.
[2]程志.从砷剂治疗白血病获效探索抗肿瘤中药的研究方向[J].浙江中医杂志,2005,3：98-99.
[3]Wyllic, A.H, Kerr, J.F.R, Curric, AR. Cell death:the significance of apoptosis[J]. Int Rev Cytol,1980,68:251-256.
[4]Lieberthal W, Levine J.S. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury[J]. Am J Physiol,1996,271:F477-12.
[5]Richard S. H, Andreas S, Jonathan E. M, et al. Mechanisms of disease cell death[J]. N Engl J Med,2009,361:1570-1583.
[6]谢新生,张秀丽,赵家军等.穿山甲煎液诱导HL 260细胞凋亡的研究[J].浙江中西医结合杂志,2001,11(8)：477-479.
[7]郭利华,李 艺.李斯文教授运用穿山甲治疗血液病的经验[J].云南中医中药杂志,2009,30(11)：1-2.
[8]张东伟,付 敏,彭贤文等.猪蹄甲与穿山甲抗小鼠骨髓微循环障碍作用的实验研究.中草药,2005,36(9)：1364-1367.
[9]许重远,李亦蕾,陈振德等.高效毛细管蛋白电泳法对鸡内金和穿山甲的鉴别[J].解放军药学学报,2007,23(6)：464.
[10]Kwong YL,Todd D.Dclicious poison:arscnic trioxide for the treatment of leukemia[J].Blood,1997,89(9):3487.
[11]Zhang P. The use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia[J]. J Biol Regul Homeost Agents,1999,13(4):195-200.
[12]Thomas X, Troncy J. Arsenic:a beneficial therapeutic poison-a historical overview[J]. Adler Mus Bull.2009 Jun;35(1):3-13.
[13]Wang H.Combined effect of docetaxel and cisplatin for non-small cell lung cancer cell lines in vitro[J].Nagoya Med Sci,2000,63(3-4):129-137.
[14]陈志炉,史亦谦,周郁鸿.中药诱导白血病细胞凋亡的研究近况[J].实用中西医结 合临床2007,7(3)：91-92.
[15]陈婷梅,祝彼得.抗白血病中药及天然药物的研究现状[J].中国中西医结合杂志,1995,15(5)：317-320.
[16]Zhang XW, Yan XJ, Zhou ZR, et al. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML[J].Science.2010,328(5975): 240-243.
[17]Cheng XD, Hou CH, Zhang XJ, et al. Effects of Huangqi (Hex) on inducing cell differentiation and cell death in K562 and HEL cells[J].Acta Biochim Biophys Sin (Shanghai).2004 Mar;36(3):211-217.
[18]于淼,李娜,胡丽娜等.动物药整理研究——穿山甲[J].吉林中医药,2009,29(6)：514-516.
[19]盂庆常.穿山甲治疗白细胞减少症应用体会[J].甘肃中医,2009,22(8)：27.
[20]Guo ZM, Li HJ, Qian XH. Gamma-globin synthesis in K562 cells induced with Tortois plastron, Astragali, Salviae miltiorrhizae and Codonopsis pilosulae[J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi,2008 Jun;16(3):520-524.
[1]Ren R.Mechanisms of BCR-ABL in the pathogeniesis of chronic myelogenous leukemia [J].Nature Reviews Cancer,2005,5 (3):172-183.
[2]高莉,李晓明.酪氨酸激酶抑制剂治疗慢性粒细胞白血病的研究进展[J].中国药房,2008,19(11)：865-867.
[3]Gora-Tybor J, Robak T.Targeted drugs in chronic myeloid leukemia [J].Curr Med Chem,2008,15 (29):3036-3051.
[4]Hehlmann R, Saussele S.Treatment of chronic myeloid leukemia in blast crisis Haematologica[J].2008,93(12):1765-1769.
[5]牛家华,王春.伊马替尼耐药慢性粒细胞白血病治疗进展[J].医药专论-世界临床药物,2008,29(5)：285-288.
[6]刘 鹤,陈晓光.慢性粒细胞白血病治疗中克服伊马替尼耐药的新策略[J].中华肿瘤杂志,2006,28(8)：561-563.
[7]邓雪蓉,王文生.慢性粒细胞白血病对STI571耐药机制的研究进展[J].白血病·淋巴瘤,2005,14(6)：382-384.
[8]PABLO RAMIREZ, JOHN F. DIPERSIO. Therapy Options in Imatinib Failures[J]. The Oncologist,2008,13:424-434.
[9]David Frame. New strategies in controlling drug resistance in chronic myeloid leukemia[J]. Am J Health-Syst Pharm,2007,64 (15):16-21.
[10]Weisberg E, Manley PW, Cowan-Jacob SW, et al. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia[J]. Nat.Rev.Cancer,2007,7:345-356.
[11]Jabbour E, Soverini S. Understanding the role of mutations in therapeutic decision making for chronic myeloid leukemia[J]. Semin Hematol,2009,46(2 Suppl 3):S22-6.
[12]Gisella Volpe, Cristina Panuzzo,Stefano Ulisciani, et al. Imatinib resistance in CML[J].Cancer Letters,2009,274:1-9.
[13]Hochhaus A,Kantarjian HM, Baccarani M, et al.Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy [J].Blood,2007,109:2303-2309.
[14]Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia:an up-date of concepts and management rec-ommendations of European Leukemia Net[J].JClin Oncol.2009,27(35):6041-6051.
[15]H.M. Kantarjian, F. Giles, N. Gattermann, et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and Intolerance [J]. Blood,2007,110:3540-3546.
[16]Michael Steinberg, Pharm D, BCOP. Dasatinib:A Tyrosine Kinase Inhibitor for the Treatment of Chronic Myelogenous Leukemia and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia[J]. Clin Ther,2007,11(29):2289-2308.
[17]ELIAS JABBOUR, JORGE E. CORTES, HAGOP M. KANTARJIAN, Suboptimal Response to or Failure of Imatinib Treatment for Chronic Myeloid Leukemia:What Is the Optimal Strategy?[J].Mayo Clin Proc,2009,84(2):161-169.
[18]Elias Jabbour, Jorge Cortes,Hagop Kantarjian.Treatment selection after imatinib resistance in chronic myeloid leukemia[J].Targ Oncol,2009,4:3-10.
[19]Michael Brave,Vicki Goodman,Edvardas Kaminskas, et al. Sprycel for Chronic Myeloid Leukemia and Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Resistant to or Intolerant of Imatinib Mesylate[J]. Clin Cancer Res,2008,14(2):352-359.
[20]Shah NP, Kantarjian HM, Kim DW, et al. Intermittent target inhibition with dasatinib 100mg once daily preserves efficacy and improves tolerability in imatinib-resistant and intolerant chronic-phase chronic myeloid leukemia[J]. JClinOncol,2008,26(19):3204-3212.
[21]O'Hare T, Walters DK, Stoffregen EP, et al. In vitro activity of BCR-ABL inhibitors AMN107 and BMS-354825 against clinically relevant imatinib resistant Ab1 kinase domain mutants[J].Cancer Res,2005,65:4500-4505.
[22]Michael W. Deininger.nilotinib[J].Clin Cancer Res,2008,4(13):4027-4031.
[23]Kantarjian HM, Giles F, Gatterman N. Nilotinib (formerlyAMN107), a highly selective BCR-ABL tyrosine kinase inhibitor,is effective in patients with Philadelphia chromosome-positivechronic myelogenous leukemia in chronic phase following matinib resistance and intolerance [J]. Blood,2007,110:3540-3546.
[24]Giles FJ, Larson RA, Kantarjian HM, et al. Nilotinib in patients with Philadelphia hromosome-positive chronic myelogenous leukemia in blast crisis who are resistant or intolerant to imatinib(abstract 1025) [J]. Blood,2007,110:310a.
[25]le Coutre P, Ottmann OG, Giles F, et al.Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is active in patients with imatinib-resistant or intolerant accelerated-phase chronic myelogenous leukemia[J]. Blood, 2008,111:1834-1839.
[26]Hughes T, Saglio G, Martinelli G, et al. Responses and disease progression in CML-CP patients treated with nilotinib after imatinib failure appear to be affected by the BCR-ABL mutation status and types [abstract 320] [J]. Blood,2007, 110(11):101a.
[27]DavidL.DeRemer,PharmD,Celalettin Ustun,Kavita Natarajan. Nilotinib:A Second-Generation Tyrosine Kinase Inhibitor for the Treatment of Chronic Myelogenous.Leukemia[J].Clinical Theapeutics,2008,30(11):1956-1975.
[28]Jabbour E, Hochhaus A, Cortes J, et al. Choosing the best treatment strategy for chronic myeloid leukemia patients resistant to imatinib:weighing the efficacy and safety of individual drugs with BCR-ABL mutations and patient history [J].Leukemia.2010,24(1):6-12.
[29]Soverini S, Colarossi S, Gnani A, et al. Resistance to dasatinib in Philadelphia-positive leukemia patients and the presence or the selection of mutations at residues 315 and 317 in the BCR-ABL kinase domain[J].Haematologica.2007,92 (3):401-404.
[30]Shah NP, Skaggs BJ, Branford S, et al.Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency[J].J Clin Invest.2007,117(9):2562-2569.
[31]Khorashad JS, Milojkovic D, Mehta P, et al. In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib[J]. Blood, 2008,111(4):2378-2381.
[32]Shah NP, Kantarjian HM, Kim DW, et al. Intermittent target inhibition with dasatinib 100 mg once daily preserves efficacy and improves tolerability in imatinib-resistant and-intolerant chronic-phase chronic myeloid leukemia[J]. J Clin Oncol.2008,26(19):3204-3212.
[33]antarjian HM, Giles F, Gattermann N, et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance[J].Blood,2007,110(10):3540-3546.
[34]Hochhaus A, Muller MC, Cortes JE, et al. Dasatinib efficacy after imatinib failure by dosing schedule and baseline BCR-ABL mutation status in patients with chronic myeloid leukemia in chronic phase (CML-CP) [J].Haematologica 2008,93: 371-372.
[35]Hughes T, Saglio G, Branford S, et al.Impact of baseline BCR-ABL mutations on response to nilotinib in chronic myeloid leukemia patients in chronic phase (CML-CP) [J] J Clin Oncol,2009,27:4204-4210.
[36]S.Soverini, A. Gnani, S. Colarossi, et al.Philadelphia chromosome-positive leukemia patients who harbor imatinib-resistant mutations have a higher likelihood of developing additional mutations associated with resistance to novel tyrosine kinase inhibitors [J]. Blood,2007,110(11):101a.
[37]Philipple Coutre, Michaela Schwarz, Theo D. Kim, New Developments in Tyro- sine Kinase Inhibitor Therapy for Newly Diagnosed Chronic Myeloid Leukemia [J].Clin Cancer Res,2010,16(6):1771-1780.
[38]Natalia L. Komarova, Allen A. Katouli, Dominik Wodarz.Combination of Two but Not Three Current Targeted Drugs Can Improve Therapy of Chronic Myeloid Leukemia[J].PLoS ONE,2009,4(2):e4423(1-5).
[39]J.Cortes,T. Bruemmendorf, H. Kantarjian,et al.Efficacy and safety of bosutinib (SKI-606) among patients with chronic phase Ph+chronic myelogenous leukemia (CML) [J]. Blood,2007,110(11):311a-312a.