实时荧光pcr在白血病分子诊断中的应用研究生物化学与分子生物学专业论文.docxVIP

中文摘要突变类型，其中CE法操作繁琐、易污染、价格昂贵、灵敏度为10．20％，测序 中文摘要 突变类型，其中CE法操作繁琐、易污染、价格昂贵、灵敏度为10．20％，测序 法有类似的缺点且灵敏度更低(20—30％)，HRM法操作方便快捷、价格便宜、 灵敏度可达5％。ARMS引物法可以检测95％的NPMl突变，包括A型、B型和 D型，灵敏度高达0．01％，可用于MRD定量。综合四种方法各自的特点，考虑 到操作难易程度和仪器普及率，结合临床样品检测情况，本研究认为在临床上可 以将HRM法和ARMS引物法结合起来用于NPMl突变的筛查，并用ARMS引 物法进行后续的MRD定量。 第四章肋簖RB融合基因的克隆、筛查和定量 针对一例罕见的儿童慢性嗜酸性粒白血病(CEL)患者进行了一系列的分子 诊断。该病人的染色体核型结果为t(1；5)(q21；q33)，分子克隆和测序证明CEL细 胞带有TPM3一PDGFRB融合基因。该病人采用格列卫进行靶向治疗并迅速达到 血液学缓解，同时微小残留病定量结果表明TPM3-PDGFRB的表达也相应降低。 最后，我们给出了一个通用的实时PCR定量体系，既可用于初诊时PDGFRB融 合基因的筛查，也可用于随访时药物疗效的评价。这些研究结果将有助于儿童 CEL患者的确诊、治疗和管理。 关键词：白血病；实时PCR；融合基因；基因突变；扩增阻碍突变系统；高分辨 熔解曲线分析；微小残留病。 2 英文摘要Abstract 英文摘要 Abstract Leukemia is one of the hematological neoplasms．During the past two decades，the development of molecular biology has greatly put forward 0111understanding of the molecular mechanisms underlying leukemogenesis．It is now well—known that chromosomal abnormalities and the resultant fusion genes are the most common causative genetic events．Another important molecular aberration is gene mutations， including point mutations，insertion mutations and deletion mutations．Many fusion genes and gene mutations have emerged as prognostic ana／or predictive biomarkers for leukemia．Molecular identification of these molecular biomarkers has not only offered important insights into the mechanisms of leukemogenesis，but also in some cases led to targeted therapies，such aS imatinib for BCR-ABLl and arsenic trioxide for PML-RARA．Moreover,these biomarkers，provided they are stable during disease,- progression，could also serve as targets for monitoring of minimal residual disease (MRD)that could herald impending relapse．The major objective of this dissertation is to develop molecular methods，in particular real-time PCR(qPCR)，for rapid and accurate detection of these leukemia-related biomarkers． In chapter one，we firstly recalled the pioneers who first described and identified leukemia as an entity．Then we reviewed the molecular genetic ev