【作者】 杨东光；

【导师】 张日；

【作者基本信息】 苏州大学 ， 内科血液学， 2007， 博士

【摘要】 一、伊马替尼与亚砷酸联合应用治疗慢性粒细胞白血病的临床观察目的:评价伊马替尼（Imatinib,IM）与亚砷酸（Arsenic Trioxide,ATO）联合应用治疗慢性粒细胞白血病（Chronic myeloid leukemia,CML）的疗效及患者的耐受性。方法:20例初诊CML慢性期（chronic phase,CP）患者口服伊马替尼400mg/d,并静脉滴注ATO注射液10mg/d,连续10d后,改为每周2次。在IM治疗后中位时间为一年的期间内,观察疗效及副作用。结果:所有患者均获得了稳定的完全血液学缓解,所需中位时间为4周。IM治疗6个月,80%患者获得了主要遗传学缓解,IM治疗12个月,所有患者均获得了主要遗传学缓解,获得主要遗传学缓解的中位时间为3个月。IM治疗6个月,55%的患者获得了完全遗传学缓解,IM治疗12个月,80%的患者获得了完全遗传学缓解。IM治疗12个月的完全分子生物学缓解率是20%。在观察期内,无1例患者出现血液学和遗传学复发,无1例耐药发生。对副作用的观察表明,IM与ATO联合应用在患者中有良好的耐受性。结论:我们初步的研究结果提示,IM与ATO联合应用是一种有前途的治疗CML的方法。并有可能因ATO的引入而克服耐药的发生,且不增加IM的副作用,在患者中有良好的耐受性。二、伊马替尼耐药的慢性粒细胞白血病患者Bcr-Abl融合基因ABL激酶区点突变的检测目的:建立IM耐药患者ABL激酶区（kinase domain,KD）点突变的检测方法,研究发生IM耐药的CML患者Bcr-Abl融合基因ABLKD发生点突变的情况。方法:采集11例发生IM耐药（血液学耐药7例,遗传学耐药4例）的CML患者IM治疗前后不同时间段共计17份骨髓,采用半筑巢式扩增长片段逆转录-聚合酶链式反应（RT-PCR）的方法,扩增Bcr-Abl融合基因ABLKD周围863bp碱基,进行纯化、测序及序列同源性分析。结果:建立了伊马替尼耐药患者ABLKD点突变的检测方法。IM耐药的CML患者ABLKD总的点突变检出率为45.45%。其中,血液学耐药患者ABLKD点突变检出率为57.14%。遗传学耐药患者ABLKD点突变检出率为25%。共发现3种点突变,G250E（2例）、E255K（1例）和T315I（2例）。血液学耐药的患者,发现突变的时间最早为IM治疗后4个月,最长为IM治疗后9个月。遗传学耐药患者突变检出时间是IM治疗后12个月。所有患者IM治疗前均未检出突变。结论:发生IM耐药的CML患者,其Bcr-Abl融合基因ABLKD存在着高频率的点突变,且与IM治疗相关。通过对耐药点突变情况进行早期检测,有利于在发生耐药前进行治疗干预,为患者提供更有效的治疗选择。三、伊马替尼耐药的慢性粒细胞白血病细胞模型的建立目的:建立IM耐药的CML细胞模型。方法:将分别含有全长Bcr-Abl融合基因发生IM耐药的T315I及E255K点突变的逆转录病毒载体MIGR1p210/T315I及MIGR1p210/E255K经亚克隆、纯化、PCR鉴定后,采用逆转录病毒介导的细胞转染技术分别转染到到依赖细胞因子生长的髓系原始细胞32D细胞中,筛选出表达Bcr-Abl的细胞,然后用荧光显微镜及蛋白免疫印记等方法鉴定阳性克隆,并分离单个克隆。经实时定量PCR（real timequantitative PCR,RQ-PCR）检测各细胞株Bcr-Abl融合基因拷贝数。结果:成功建立了IM耐药的CML细胞模型32Dp210^T315I及32Dp210^E255K,两细胞株Bcr-Abl融合基因拷贝数均较高,并表达Bcr-Abl融合蛋白。结论:建立了IM耐药的CML细胞模型,为研究IM耐药的CML提供了新的有力工具。四、亚砷酸对伊马替尼耐药的慢性粒细胞白血病细胞模型的作用及机制目的:探讨ATO与IM是否存在交叉耐药现象以及ATO对Bcr-Abl阳性细胞的作用是否存在靶向性,探讨ATO与IM对IM耐药细胞株联合应用的性质,探讨ATO对IM耐药的CML细胞株的作用及其作用机制。方法:采用四氮唑蓝（methyl-thiazol-tetrazolium,MTT）法测定IM、ATO单独及联合应用于32D/MIG、32D/p210、32D/p210^T315I及32D/p210^E255K的IC₅₀。测定伊马替尼、亚砷酸单独及联合应用于32D/p210及32D/p210^E255K的IC₈₀,计算联合应用指数。通过流式细胞仪检测ATO对32D/p210、32D/p210^T315I及32D/p210^E255K细胞凋亡及免疫印记检测ATO对HCK/STAT信号转导通路的影响。结果:IM对32D/MIG、32D/p210、32D/p210^T315I及32D/p210^E255K的IC₅₀分别为＞10μM、0.17±0.02μM、＞10μM及3.31±6.40μM。而ATO对上述细胞株的IC₅₀分别为0.58±0.03μM、0.35±0.05μM、0.38±0.04μM及0.41±0.06μM。两者以固定比例联合应用时,对32D/p210的联合应用指数分别为CI:IC₅₀:1.10±0.07、CI:IC₈₀:0.88±0.20,对32D/p210E255K的联合应用指数分别为CI:IC₅₀:1.00±0.07、CI:IC₈₀:0.90±0.31。ATO对32D/p210、32D/p210^T315I及32D/p210^E255K诱导的凋亡表现出时间-剂量依赖性。ATO作用于32D/p210、32D/p210^T315I及32D/p210^E255K48h时,对HCK/STAT途径有显著的抑制作用。结论:IM对32D/p210^T315I及32D/p210^E255K耐药,ATO与IM无交叉耐药现象。ATO对Bcr-Abl阳性细胞株的治疗表现出靶向性。在IM自身能克服耐药的浓度下,ATO与IM对IM耐药的细胞株表现为相加或协同作用。ATO对IM耐药的CML细胞株的作用与其诱导凋亡和下调HCK/STAT信号转导通路有关。五、亚砷酸对慢性粒细胞白血病细胞株裸鼠皮下成瘤的抑制作用及机制目的:探讨ATO对裸鼠32D/p210移植瘤的抗肿瘤作用及机制,并探讨ATO的毒副作用。方法:建立32D/p210移植瘤模型,随机分为3组,ATO高、低剂量组分别给予ATO 5、2.5mg/kg/d,连续10天,腹腔注射;生理盐水组给予等体积的生理盐水,用法同ATO组。观察不同浓度ATO治疗组及对照组瘤块大小、抑瘤率,取瘤组织行病理学检查和瘤细胞超微结构观察,并对治疗后瘤组织的Bcr-Abl融合基因拷贝数进行测定。观察裸鼠一般状态的变化,并在光镜下观察裸鼠肝、肾结构的改变,同时对裸鼠进行治疗后血常规的检测。结果:ATO高、低剂量组的瘤组织大小均较生理盐水组明显缩小;瘤组织病理可见ATO高剂量组以中度坏死为主,低剂量以轻度坏死为主。电镜下发现ATO高、低剂量组瘤组织的细胞出现凋亡。经治疗,Bcr-Abl融合基因拷贝数显著降低,高剂量组拷贝数的降低明显大于低剂量组（P＜0.0001）。ATO高、低剂量组裸鼠一般状态良好,ATO无明显的肝、肾毒性,对血象也无明显影响。肾脏病理仅在高剂量组出现了轻微的肾小管上皮细胞水肿和灶性蛋白管型,仅轻微的降低了血红蛋白,且高剂量组降低的较为明显。结论:32D/p210对裸鼠具有高致瘤性,ATO对该瘤有明显抑制作用,并能显著降低Bcr-Abl融合基因拷贝数,其作用呈明显的剂量依赖性,其作用机制与诱导凋亡有关。ATO对裸鼠的肝、肾和造血系统无明显的副作用。更多还原

【Abstract】 PartⅠ:Clinical Observation of Combination Therapy of lmatinib With Arsenic Trioxide for Chronic Myeloid Leukemia Patients.Objective:To evaluate the effect of combination therapy of Imatinib（IM） With Arsenic Trioxide（ATO） for Chronic Myeloid Leukemia（CML） patients and patients’ tolerance of this therapy.Methods:ATO was administered via intravenously guttae at a dose of 10mg for one week,then twice a week,along with oral IM 400mg daily to 20 CML patients in chronic phase（CP）.The effect and adverse effect of this therapy was observed after a median time of one year.Results:All the patients achieved and maintained a complete hematologic response after a median time of 4 weeks.In the sixth month,the major cytogenetic response rate is 80%.In the twelveth month,all the patients achieved major cytogenetic response.The median time to achieve major cytogenetic response is 3 months.In the sixth month,55%of the patients achieved major cytogenetic response.In the twelveth month,80%of the patients achieved complete cytogenetic response.In the twelveth month,the complete molecular response is 20%.No one relapsed in the 12 months’ observation.It seems that this therapy can be tolerated well in patients.Conclusions:Our preliminary result indicates that the combination of IM and trisenox to treat chronic myeloid leukemia is a hopeful therapy and may be promising in avoiding the occur of IM resistance. PartⅡ:Examination of Point Mutations Within the ABL Kinase Domain of Bcr-Abl Fusion Gene in Patients with Chronic Myeloid Leukemia Who Develop Imatinib ResistanceObjective:To establish a method for investigating the point mutations within the ABL kinase domain（KD） of Bcr-Abl fusion gene in patients with chronic myeloid leukemia who develop IM resistance and investigate the mutation.Method:We collect a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance（n=7） or cytogenetic refractoriness（n=4）.A long semi-nest PCR method was used to amplify the ABL KD of the Bcr-Abl fusion gene. After two cycles of PCR reaction,we get a fragment including 863 bases,purified and sequenced it.Result:We have established the method for investigating the point mutations within the ABL KD of Bcr-Abl fusion gene in patients with chronic myeloid leukemia who develop IM resistance.The ABL point mutation was detected in 45.45%of patients.The mutation rate of hematology resistance is 57.14%and the mutation rate of cytogenetic refractoriness is 25%.In total,we find three point mutation in all of our patients tested,ie,G250E（2）,E255K（1） and T315I（2）.In patients who develop hematology resistance,the earliest time to detecte mutation is 4 months later after the therapy of IM and the longest time to detecte mutation is 9 months later after the therapy of IM.In patients who develop cytogenetic refractoriness,the time to detecte mutation is 12 months later after the therapy of IM.For the patients whose samples are available,no single mutation happens before IM thearpy.Conclusion:There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of Bcr-Abl fusion gene in patients with chronic myeloid leukemia,and the mutation is related to IM therapy.It’s good for patients to switch to another therapeutic strategy when the mutations were detected earlier.PartⅢ:Establishment of the Chronic Myeloid Leukemia Cells Models which is Resistant to IMObjective:To establish the chronic myeloid leukemia cell models which are resistant to IM.Method:After subcloned,purified and PCR verificated the retroviral vectors which comprise the resistance point mutations full length bcr-abl fusion gene,namely, MIGRlp210/T315I and MIGRlp210/E255K,we transfected them to IL-3 dependent myeloid initial cell 32D respectly.We secreened the positive clones and verified them with fluorescent microscope and immune blot.Then we select the monoclone.And we also detect the copies of b3a2 Bcr-Abl fusion genes by real time quantitative PCR（RQ-PCR）.Result:We have established the chronic myeloid leukemia cell models which are resistant to IM,namely,32Dp210^T315Iand 32Dp210^E255K),and the copies of Bcr-Abl fusion genes of which are high and Bcr-Abl fusion protein of which are expressed.Conclusion:We have established the chronic myeloid leukemia cell models which are resistant to IM,and they will become a powerful tool to investigate the chronic myeloid leukemia which is resistant to IM.PartⅥThe Action of Arsenic Trioxide to the Chronic Myeloid Leukemia Cell Models which are Resistant to Imatinib and its MechanismObjective:To study the whether IM-resistant cell lines induce cross-resistance to ATO,and whether ATO is a targeted drug for Bcr-Abl positive cells.And also we investigate the action of ATO to the chronic myeloid leukemia cell models which are resistant to IM and its mechanism.Methods:The methyl-thiazol-tetrazolium（MTT） assay was performed to determine the 50%inhibition of proliferation(IC₅₀) of ATO,IM and their combination to 32D/MIG;32D/p210,32D/p210^T315I) and 32D/p210^E255K.And the 80%inhibition of proliferation(IC₈₀) of ATO,IM and their combination to 32D/p210 and 32D/p210^E255K are also determined to calculate the combination index.In addition,we determine the effect of ATO to 32D/p210,32D/p210^T315I and 32D/p210^E255K on apoptosis by flow cytometer and on HCK/STAT signal transduction pathway by immune blot.Results:The IC₅₀ of IM to 32D/MIG;32D/p210,32D/p210^T315I and 32D/p210^E255K are＞10μM,0.17±0.02μM,＞10μM and 3.31±6.40μM,respectly.While the IC₅₀ of ATO to cells above is 0.58±0.03,0.35±0.05,0.38±0.04 and 0.41±0.06μM.When they combined in a fixed ration,their combination index to 32D/p210 is that,CI:IC⁵⁰ is 1.10±0.07 and CI:IC⁸⁰ is 0.88±0.20,and their combination index to 32D/p210E255K is that,CI:IC₅₀ is 1.00±0.07 and CI:IC₈₀ is 0.90±0.31.The inducing apotosis of ATO to 32D/p210,32D/p210^T315I and 32D/p210^E255K shows time and dose dependent way.ATO can significantly downregulate HCK/STAT pathway when they acts on 32D/p210, 32D/p210（T315I） and 32D/p210^E255K for 48 hours.Conclusion:IM is resistant to 32D/p210^T315Iand 32D/p210^E255K,while there is on cross-resistence of ATO to IM.ATO is a targeted drug to Bcr-Abl positive cell lines. Only in a concertration when IM can overcome resistance,ATO and imatininb shows additive or synnergism.The main mechanism of ATO acts on Bcr-Abl positive cells related to its inducing apotosis and deregulating HCK/STAT signal transduction pathway.PartⅤThe Studies on Anti-tumor Effect and Its Mechanism of Arsenic Trioxide for Chronic Myeloid Leukemia Cell Line in Nude MouseObjective:To study the effect of ATO on 32D/p210 in vivo,as well as the related mechanism and toxicity.Methods:The transplantable 32D/p210 tumor models in nude mice were established and were divided into 3 groups at random.ATO were injected imtraperitoneally into nude mouse at a dose of 2.5mg/kg and 5mg/kg respectively on arsenic trioxid high dose and low dose group.And the saline was given to the NS group in the equal volume and fasion.We studied the change of the size of tumor and general condition of nude mouse.The tumor tissues were examined by pathology and electron microscope and the copies of Bcr-Abl fusion gene is measured after treatment.We observed the structural change of liver and kidney and the hematology regular test.Results:Compared with NS group,the tumor volume and weight in each group of ATO was reduced significantly（P＜0.05）.Slight necrosis was found in tumor tissues of ATO 2.5mg/kg,and moderate necrosis was found in tumor tissues of ATO 2.5mg/kg. Apoptosis characteristics could be seen in tissues of ATO groups under electron microscope.The copies of Bcr-Abl fusion gene is significantly decreased,and the decreases is more obviously in the ATO 5mg/kg group.There is no side effect to the liver, kidney and the system of blood,except for slight renal tubular epithelial cellular edema and focus protein cast and decreases in hemoglobin,which is significant in ATO 5mg/kg.Conclusion:32D/p210 has the high tumorigenicity in nude mice and ATO injection has a significant anti-tumor effect on nude mice model,which may be related to inducing the apoptosis of cell,and the effect shows dose dependent.ATO has no obvious injury for hepatic,renal and hematopoietic system of the nude mice.更多还原