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BMI-1及HOXA9基因在儿童急性白血病外周血中的表达及临床意义

BMI-1 and the HOXA9 Gene in Children with Acute Leukemia Peripheral Blood and Their Clinical Significance

作者: 专业:儿科学 导师:王军 年度:2010  院校: 郑州大学

Keywords

        白血病是造血系统的恶性疾病,是国内十大高发恶性肿瘤之一,其特点为造血组织中某一类型的白血病细胞在骨髓或其他造血组织中恶性增生,并浸润体内各脏器、组织,导致正常造血细胞受抑制,产生各种症状。急性白血病中一部分病例因化疗耐药,效果不佳,并发症多者预后较差。随着医学的发展与进步,急性白血病的治疗水平也有了很大提高,积极预防治疗并发症,提高机体抗病能力,可以挽救患者生命。现在,白血病的治疗方法有化疗、中西医结合治疗、骨髓移植、生物调节,而目前基因治疗白血病是研究领域的难点和热点之一。我国白血病患者约为3-4人/10万人口,小儿的恶性肿瘤中以白血病的发病率最高。由于白血病的发病机制尚未完全明确,困扰着临床治疗,因此阐明白血病的发病机制,进一步研究基因在儿童白血病发病机制中的作用,应用基因来达到治疗目的是一条新的途径。原癌基因BMI-1(B-cell specific moloney leukemia virus insertion site 1, BMI-1)属于PcG (Polycomb group, PcG)家族中PRCI复合体系,与其他各种PcG蛋白一起通过形成多蛋白复合体参与同源盒基因(homobox, HOX)的转录调节,维持HOX的抑制状态。HOX在决定细胞定向分化与增殖,调控机体组织器官发育方面起决定性作用,BMI-1基因表达异常导致HOX基因表达抑制或激活异常,影响HOX基因正常功能的发挥。PcG介导的基因沉默是局部事件,不会影响较大范围的染色质区域,调节基因表达的中心环节是BMI-1,其他的PcG蛋白影响很小。研究提示PcG蛋白家族中的BMI-1和Ring1B是PRC1复合体的核心亚单位,该复合体在调节HOX基因表达、X染色体失活、肿瘤发生和干细胞自我更新中发挥重要作用。越来越多的证据表明,HOX基因在白血病的形成中起到了重要的作用。HOX家族基因广泛的分为两大类:HOX基因和non—HOX基因。HOX家族中HOXA9、HOXA10、HOXB3、HOXB4、HOXB6在许多急性髓系白血病(acute myelogenous leukemia, AML)患者中常可检测到高水平的表达。HOXA9在AML中表达较为普遍,除FAB分型为M3型的AML外均高度表达,在慢性粒细胞白血病(chronic myeloegenous leukemia, CML)和骨髓增生异常综合征-原始细胞增多型(myelodysplastic syndrome, MDS-RAEB)中也有表达。在24个白血病细胞系中,8个髓系白血病细胞株除HL60、TMM外均可检测到HOXA9的表达,HOXA9在鼠骨髓细胞中无限制的表达3-10个月后,将不可避免的导致白血病的发生,提示AML发生前已有遗传学的改变。BMI-1基因可维持干细胞池,是成体干细胞及白血病干细胞自我更新所必需的基因之一,作为致癌基因,BMI-1与造血系统恶性肿瘤及实体瘤的发生、发展密切相关。BMI-1基因有望成为一种用于恶性肿瘤的诊断、病情评估、患者预后和判断恶性肿瘤进展的新指标之一。BMI-1是HOX基因高度特异的静止子,将基因导入淋巴细胞白血病或淋巴瘤细胞中,可下调淋巴祖细胞中HOX靶基因。近几年的研究发现BMI-1和/或HOX在多种肿瘤高表达,提示BMI-1/HOX在研究肿瘤发病机制和治疗等方面可能具有较高的潜在价值。BMI-1作为一个在多种肿瘤中异常表达的癌基因,随着基因治疗研究的进展,在未来基因治疗人类肿瘤上的应用前景将是十分广阔的。人们设想使BMI-1基因和/或HOX基因增加和/或缺失,作为靶点来治疗由上游或者下游基因变化引发的白血病。但目前关于BMI-1在肿瘤细胞中过表达或下调后对HOX基因表达变化的影响的研究还很少。由此设计该实验,观察BMI-1mRNA、HOXA9mRNA在儿童急性白血病外周血及健康儿童外周血中的表达状况和两者间的相关性及其相互影响,进一步探讨白血病的发病机理,为以后白血病的基因治疗提供理论依据。目的1.观察BMI-1及HOXA9基因在儿童急性白血病(acute leukemia, AL)外周血中的表达情况及在AL发生中的临床意义。2.探讨AL中BMI-1与HOXA9基因两者间的相关性。3.探讨白血病的发病机理,为以后白血病的基因治疗提供依据。材料与方法标本取自2008年6月至2009年6月郑州大学第三附属医院和郑州市其他医院诊治的急性白血病儿童及健康儿童的外周血。初治患者经联合化疗第19天复查骨髓象判断是否完全缓解(CR)。其诊断、CR及复发的判断标准均符合2006年中华医学会修订的急性白血病诊断标准。急性白血病儿童标本57例,男女比例为35:22(1.59:1),年龄3个月~14岁,中位年龄5岁。其中急性淋巴细胞性白血病(ALL)38例(B型27例;T型11例),急性髓性白血病(AML)19例(M13例、M25例;M36例;M42例;M53例),经过骨髓细胞形态学、流式细胞免疫表型、染色体等检查确诊。以年龄3个月-12岁,中位年龄6岁健康儿童30例为对照组,男(18例):女(12例),比例为:1.50:1。各组年龄、性别比较差异无统计学意义。运用半定量逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法检测其外周血BMI-1和HOXA9基因的表达情况,并追踪随访上述基因阳性及阴性患者的缓解率及生存期。采用SPSS13.0统计软件进行分析,所有原始数据均进行正态分布和方差齐性检验。实验数据以x±s表示,多样本均数比较用单因素方差分析,相关分析用pearson线性相关分析方法,以α=0.05为检验水准。结果1. BMI-1基因、HOXA9基因在急性白血病患儿外周血中的表达水平明显高于对照组(p<0.05)。2. BMI-1基因在B淋巴细胞性白血病外周血中均有表达,其在粒细胞性白血病外周血中部分表达或弱表达,在B淋巴细胞性白血病表达高于粒细胞性白血病(p<0.05)。3. HOXA9基因在急性髓系白血病(除AML-M3)外周血中表达较为普遍,HOXA9mRNA在急性髓系白血病中的表达水平明显高于急性淋巴细胞白血病患者,两者相比有显著统计学意义(P<0.01)。4.联合化疗未缓解组BMI-1mRNA及HOXA9mRNA表达水平高于缓解组,但两者相比差异无统计学意义(p>0.05),缓解后均未检测到BMI-1mRNA及HOXA9mRNA表达;初治组、复发组与完全缓解组相比,前两者表达显著高于后者(p<0.05);初治组、复发组与对照组相比有显著差异性(p<0.05),完全缓解组与对照组相比无显著差异性(p>0.05)。5.在研究过程中,追踪随访上述基因阳性及阴性患者的缓解率及生存期,在57例患者中共有23例患者观察到治疗情况,其中缓解19例,未缓解4例,总缓解率为85%(19/23);对其中白血病患儿HOXA9mRNA表达高低与临床疗效、生存率间进行变量直线相关分析,分析结果提示临床治疗效果、生存率与HOXA9mRNA表达呈负相关,HOXA9mRNA表达水平越高,临床治疗就越差,生存率也就越低。相关系数r=-0.857,经t检验P<0.01。6.急性白血病患儿BMI-1、HOXA9两者的表达水平进行变量线性相关分析,结果提示BMI-1与HOXA9mRNA表达呈负相关,BMI-1mRNA表达水平越高,HOXA9mRNA表达水平越低;相反BMI-1mRNA表达水平降低,HOXA9mRNA表达水平升高。相关系数r=-0.667,经t检验P<0.01。结论1. BMI-1基因具有淋系特异性,HOXA9具有髓系特异性,可作为区别淋巴细胞性白血病和髓性细胞性白血病的标志性基因之一。2. BMI-1基因、HOXA9基因亦可作为儿童急性白血病的发病、复发及治疗和预后判断的指标之一。3.原癌基因BMI-1与HOXA9表达水平与肿瘤细胞负荷有关,高水平表达急性白血病儿童治疗及预后可能较差。4. BMI-1与HOXA9在儿童AL中表达水平两者间有明显负相关性。
    Leukemia is a malignant disease of hematopoietic system, which is one of the top ten high incidence of malignant tumors, characterized by a certain type of hematopoietic tissue cells in the bone marrow of leukemia or other blood-forming tissue in the incidence of malignant hyperplasia,and infiltration in vivo every organs, organizations, leading to normal hematopoietic cells being inhibited, resulting in a variety of symptoms. Acute leukemia is a blood disease in acute and danger severe case for some cases because of resistance to chemotherapy,results are poor, many complications those who comparatively are poor prognosis.With the development and progress of medicine, acute leukemia treatment level has also been greatly improved, and an active preventive treatment for complications and improving the body’s resistance to diseases, can save patient’s life. Methods of treatment of leukemia chemotherapy, combines Chinese and Western medicine treatment, bone marrow transplant, bio-regulators and so on, and the present field of gene therapy for leukemia research, one of the difficult and hot. Leukemia patients in China is about 3~4/10 million people, childhood ’s malignant tumor is the highest incidence of leukemia. Because of the pathogenesis of leukemia has not yet entirely clear, troubled by the clinical treatment, thus to clarify the pathogenesis of leukemia, to further study the effectiveness of genes in the pathogenesis of leukemia in children, to utilize genes to achieve goals of treatment is a new way.Proto-oncogene BMI-1 (B-cell specific moloney leukemia virus insertion site 1, BMI-1) belongs to PcG (Polycomb group, PcG) family of PRCI composite system, together with other PcG proteins through the formation of multi-protein complexes involved in homeobox(homobox,HOX)gene transcription regulation, the maintenance of HOX the inhibitory state. HOX orientation in determining cell differentiation and proliferation, regulation of body tissues and organs and development play a decisive role.BMI-1 gene expression in abnormal expression lead to HOX gene inhibition or activation abnormalities, affecting the normal function of HOX genes play. Epigenetic PcG-mediated gene silencing is a local event, does not affect a broader range of chromatin regions. regulating gene expression in the central link is the BMI-1, the other PcG proteins has little effect.. Research suggests that PcG family of the BMI-1 and Ring1B is core subunits in PRC1 complex and the complex regulate HOX gene expression, X chromosome inactivation, tumorigenesis and stem cell self-renewal, to play an important role. More and more evidences suggest that, HOX genes in leukemia formation has played an important role. HOX family genes are divided into two broad categories:HOX genes and non-HOX genes. HOX family of HOXA9, HOXA10, HOXB3, HOXB4, HOXB6 in a number of acute myeloid leukemia (AML) can be detected frequently in patients with high levels of expression. HOXA9 expression in AML is more common.In addition to FAB classification of M3 type AML things are highly expressed and chronic myeloegenous leukemia. (CML) and myelodysplastic syndrome-primitive cells increased type (MDS-RAEB) also has expressed. In the 24 leukemia cell lines,eight myeloid leukemia cell lines except HL60, TMM can be detected the HOXA9 expression, HOXA9 in mouse bone marrow cells in the unrestricted expression of 3 to 10 months, will inevitably lead to the occurrence of leukemia.To hint that AML have occurred prior to genetic changes.BMI-1 gene can maintain the stem cell pool.It is one of necessary self-renewal genes for adult stem cells and leukemia stem cells, as a cancer-causing gene, BMI-1 closely correlate the development of hematopoietic malignancies and solid tumors of the genesis. BMI-1 gene is expected to be one of new indicators of progress for cancer diagnosis,disease assessment,and judge the prognosis of patients with malignant tumors.BMI-1 is highly specific static son of HOX gene,and BMI-1 gene introducted into lymphocytic leukemia or lymphoma cells,which can down-regulate HOX target genes in lymphoid progenitor cells.In recent years,the study found that BMI-1 and/or HOX highly expressed in a variety of tumors, suggesting that BMI-1/HOX may have a high potential value in the study of directions of pathogenesis and treatment and so on. BMI-1, as a cancer gene abnormally expressed in a variety of tumors, along with the progress of research into gene therapy in the future gene therapy of human tumors widelly used. It is envisaged that BMI-1 gene and/or HOX genes will be increased and/or missinged, as a target for the treatment of leukemia caused by the upstream or downstream from the genetic changes. But at present after the, There is little study of concerning HOX genes change caused by BMI-1 in tumor cells over-expression or reducing.Thus the experiment is designed to observe the BMI-1mRNA, HOXA9mRNA in children with leukemia and healthy children peripheral blood expressed in order to explore the expression of these two genes and the possible relevance of their interaction, to further explore the pathogenesis of leukemia and provide clues to gene therapy for leukemia.Objective1. To observe BMI-1 and the HOXA9 gene expression in children with acute leukemia (acute leukemia, AL) peripheral blood, and explore the clinical significance of the expression of these two genes in the AL.2. To explore the possible correlation of the BMI-1 and the HOXA9 gene in the AL peripheral blood.3. To explore the pathogenesis of leukemia, and provide clues to gene therapy for leukemia. Materials and methodsIn June 2008 to June 2009 in the Third Affiliated Hospital of Zhengzhou University and other hospitals, Zhengzhou City, diagnosis and treatment for children with acute leukemia and good health children peripheral blood are collected as samples. Combined with chemotherapy in newly diagnosed patients by the 19th day review to determine whether the bone marrow complete remission (CR). The diagnosis, CR and relapse criteria for judging are in line with the Chinese Medical Association 2006 revised diagnostic criteria of acute leukemia. Children with acute leukemia specimens of 57 cases, male to female ratio of 35:22 (1.59:1), aged 3 months to 14 years old, with a median age of 5 years old. With acute lymphoblastic leukemia (ALL) 38 cases (B-type 27 cases; T-type 11 cases), acute myeloid leukemia (AML) 19 cases (M1 3 cases, M2 5 cases; M3 6 cases; M4 2 cases; M5 3 cases), the diagnosis is confirmed after bone marrow cell morphology, flow cytometry, immunophenotype, chromosomes. By age 3 months to 12 years old, with a median age of 6 years of good health 30 cases of children in the control group, male (18 cases):female (12 cases) ratio:(1.50:1). Of all age groups, sex difference was not statistically significant. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method is used to detect 30 cases of good health of children and 57 cases of childhood AL in patients with BMI-1 and HOXA9 gene expression and to track follow-up of the above-mentioned gene positive and negative patients with remission rates and survival. SPSS 13.0 statistical software is used for analysis, all the raw data are for normal distribution and test of homogeneity of variance. Experimental data indicate by x±s comparision of a variety of the mean is with the single-factor analysis of variance, correlation analysis uses linear correlation pearson analysis, a=0.05 level for the test.Results1. The level of BMI-1/HOXA9 gene’expression in children with acute leukemia peripheral blood was significantly higher than that of control group (p<0.05).2. We found part of the BMI-1 gene expression or weak expression in children with myelogenous leukemia peripheral blood, while universal expression was found in various types of B-lymphocytic leukemia, and higher than that in myelocytic leukemia group (p<0.05).3.The children in the HOXA9 gene expression in acute myeloid leukemia peripheral blood is more common, HOXA9 mRNA expression was significantly higher than in children with acute lymphoblastic leukemia patients,the comparision of the two statistically significant (P<0.01).4. The level of expression in chemotherapy non-remission groups was higher than that in remission group, but the difference was not statistically significant(p>0.05).After complete remission,BMI-1mRNA/HOXA9 mRNA were not detected in the two groups;Compared with the complete remission group,expression of BMI-1mRNA/HOXA9 mRNA in the untreated group and the recurrence group was significantly higher (p<0.05);and expression of BMI-1mRNA/HOXA9 mRNA in the former two was significantly different from that in the control group(p<0.05);while there was no significant difference between the complete remission group and the control group(p>0.05).5. In the research,to track follow-up of remission rates and survival of the above-mentioned gene positive and negative patients.we observed 23 cases children with acute leukemia specimens of 57 ones, remission ratio is 0.85 (19/23).HOXA9 gene expression these whose level was carry out a variable linear correlation analysis. results suggest that clinical outcomes/survival and HOXA9mRNA expression was negatively correlated.concerned with tumor cell load,high level expression those who were most poor clinical outcomes and prognosis, the expression levels of HOXA9mRNA were significantly inversely correlated with clinical outcomes and survival(r=-0.857,P<0.01).6. The levels of BMI-1 and HOXA9 gene’expression in children with acute leukemia was carry out a variable linear correlation analysis, results suggest that BMI-1 and HOXA9 mRNA expression was negatively correlated, The higher expression level of BMI-1mRNA, the lower expression levels of HOXA9mRNA;on the contrary BMI-1mRNA reduce the level of expression, HOXA9mRNA increased expression levels. The correlation coefficient r=-0.667,by t test P<0.01. Conclusion1. BMI-1 gene has a lymphoid lineage-specific,HOXA9 has a myeloid lineage-specific, can serve as a distinction between lymphoblastic leukemia and myelogenous leukemia one of gene symbol.2. BMI-1 gene and HOXA9 gene may be used as one of indicators of the incidence of children with acute leukemia, relapse and treatment and prognosis.3.The HOXA9 expression level is related to tumor cell burden,the higher levels of expression, the poor treatment and prognosis poor.4. BMI-1 and HOXA9 expression levels have significantly negative correlation between the two.
        

BMI-1及HOXA9基因在儿童急性白血病外周血中的表达及临床意义

摘要4-8
Abstract8-13
中英文缩略词对照表14-16
正文部分16-41
    引言16-18
    材料与方法18-24
    结果24-29
    讨论29-38
    参考文献38-41
综述部分 原癌基因BMI-1与HOX基因在儿童白血病中相关研究41-62
    参考文献57-62
个人简历 在校期间发表的学术论文及成果62-63
致谢63
        下载全文需63


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