XIAP及其相关因子1在急性淋巴细胞白血病中表达意义

目的:探讨X染色体连锁凋亡抑制蛋白(XIAP)及其相关因子(XAF1)在急性淋巴细胞白血病(ALL)中的表达及其临床意义,并评估其在临床治疗及预后中的价值。方法:采用病例对照研究,应用实时荧光定量聚合酶链反应(RQ-PCR)检测85例ALL患者骨髓标本中XIAP及XAF1的mRNA表达水平。结果:XIAP mRNA表达水平初诊ALL组高于CR组和对照组(P<0.05),而低于复发组(P<0.05),CR组表达水平高于对照组(P<0.05);而XAF1在ALL时呈低表达或不表达,CR组表达高于ALL其它组(P<0.05),与对照组差异无统计学意义(P>0.05)。XIAP及XAF1二基因表达水平在T系ALL与B系ALL,成人与儿童,男女性别之间表达水平差异无统计学意义(P>0.05)。XIAP/XAF1比值在ALL患者中初诊组和复发组明显高于对照组和缓解组(P<0.05),缓解组高于对照组(P<0.05)。结论:ALL患者XIAP基因高表达,而XAF1呈现低表达或不表达,提示XIAP可能通过抑制白血病细胞凋亡参与了ALL的发生发展,并与预后不良及治疗反应相关。ALL中XIAP与XAF1表达水平的不平衡,可能是ALL预后不良及复发的一项重要因素之一。抑制XIAP及上调XAF1基因来治疗ALL,将为ALL的基因治疗提供新思路。     

38例不同病期慢性粒细胞白血病患者bcr-abl基因的表达

目的 探讨慢性粒细胞白血病(CML)不同病期bcr-abl基因的表达。方法 应用RQ-PCR方法检测38例共45份不同临床分期CML病人外周血中bcr-abI的表达水平。结果 CML病人的bcr-abl基因和bcr-abl/abl比率在慢性期分别为(11542±5106)拷贝/μg总RNA和(10.58±5.12)％;加速期分别为(83350±7844)拷贝/μg总RNA和(84.20±3.78)％;急变期分别为(79112±7956)拷贝/μg总RNA和(80.15±4.16)％。bcr-abl基因表达水平与CML不同临床病期密切相关。结论 RQ-PCR检测bcr-abl基因表达可作为CML疾病进展、预后判断和骨髓移植后微小残留疾病监测的良好指标。     

RQ-PCR检测Ig/TCR基因重排监测急性淋巴细胞白血病患儿治疗过程微小残留白血病

目的 研究RQ-PCR检测急性淋巴细胞白血病（ALL）患儿Ig/TCR基因重排在微小残留白血病（MRD）监测中的作用。方法 以2009年3月至2011年3月在广州市妇女儿童医疗中心血液肿瘤科确诊和治疗的ALL患儿为研究对象,PCR检测初诊ALL患儿的Ig/TCR基因重排;基因扫描分析初诊患儿Ig/TCR基因重排的克隆特性;对ALL患儿的单克隆性Ig/TCR基因重排进行测序,RQ-PCR检测不同治疗阶段Ig/TCR基因重排的表达量。结果 86例ALL患儿进入分析,男52例,女34例;年龄1~13（4.3±3.0）岁,随访时间1~26（14.3±7.0）个月。①83例（96.5%）检出1种或以上Ig/TCR基因重排,共检出209个Ig/TCR基因重排;②91.8%（56/61例）检出1种或以上单克隆性Ig/TCR基因重排;61例172个Ig/TCR基因重排中,单克隆性、寡克隆性和多克隆性Ig/TCR基因重排的检出率分别为58.1%（100个）、30.8%（52个）和11.0%（19个）,差异有统计学意义（P=0.000）;③26例完成连续3次随访,其中22例持续完全缓解患儿的Ig/TCR基因重排平均相对表达量持续下降,在维持治疗前均为MRD阴性（≤1.0×10-4）;4例复发患儿在诱导缓解治疗后至复发前各检测时点Ig/TCR基因重排表达量均＞1.0×10-4,并在复发前已有回升,从开始回升至临床复发的平均时间为3.75（2～8）个月。结论 Ig/TCR基因重排相对表达量可反映MRD水平,可作为判断预后、监测复发和指导治疗的有效手段。     

BCR/ABL重组质粒实时荧光定量PCR标准品的制备

目的：用T-A克隆法构建含BCR/ABL融合基因的重组质粒,并用实时定量PCR（RQ-PCR）方法制备标准品。方法：通过培养细胞,提取总RNA并逆转录为cDNA后做PCR,电泳胶回收纯化,T-A克隆与pUCm-T载体连接,转染DH5a菌,蓝白斑筛选阳性菌落后,大量提取质粒,再进行RQ-PCR,最后制得BCR/ABL的重组质粒标准品。结果：蓝白斑筛选实验、PCR扩增均证实BCR/ABL融合基因重组到pUCm-T载体上,经RQ-PCR定量后得到BCR/ABL重组质粒标准品的标准曲线。结论：该方法能大量制备质粒标准品,并且可被推广应用。     

Impact of two independent bone marrow samples on minimal residual disease monitoring in childhood acute lymphoblastic leukaemia

Minimal residual disease (MRD) diagnostics are used for risk group stratification in several acute lymphoblastic leukaemia (ALL) treatment protocols. It is, however, unclear whether MRD is homogeneously distributed within the bone marrow (BM) and whether this affects MRD diagnostics. We, therefore, analysed MRD levels in 141 paired BM samples (two independent punctures at different locations) from 26 ALL patients by real-time quantitative polymerase chain reaction (PCR) analysis of immunoglobulin and T-cell receptor gene rearrangements. MRD levels were comparable in 112 paired samples (79%), whereas two samples (both taken at day 15) had MRD levels that differed more than threefold. In the remaining 27 paired samples, MRD could be quantified or detected in one sample only. In four patients, MRD-based risk group classification was dependent on the site of BM puncture. Repetition of MRD analyses using 10-fold replicates instead of triplicates resolved most differences. In conclusion, MRD levels in paired BM samples were highly comparable, indicating that it is sufficient to analyse MRD in a single sample only. Nevertheless, MRD-based risk group classification can differ between paired BM samples, mainly because of variation below the quantitative range of the PCR assay rather than to a different distribution of leukaemic cells within the BM.     

实时定量PCR监测B细胞恶性肿瘤患者造血干细胞移植后的IgH水平及意义

本研究评价实时定量PCR(RQ-PCR)技术检测IgH水平对B细胞恶性肿瘤患者造血干细胞移植(HSCT)后残留肿瘤细胞监测的意义.采用家族一致性TaqMan探针联合等位基因特异性寡核苷酸(ASO)上游引物技术检测22例B细胞恶性肿瘤患者HSCT前后骨髓单个核细胞的IgH水平动态变化.IgH水平以内参基因GAPDH进行归一化.结果表明,RQ-PCR实验可重复灵敏度为1个拷贝.9例IgH单克隆重排患者,在HSCT后1个月骨髓中IgH的拷贝数较初治时明显降低(6.67×103/106 GAPDH vs 29/106 GAPDH,p＜0.01).3例移植后15个月IgH的拷贝数持续小于102/106 GAPDH,18个月后IgH水平为0的患者获得了完全的临床和分子遗传学缓解(CCyR);5例移植后3个月以内IgH拷贝数持续小于102/106 GAPDH,随后IgH拷贝数持续小于103/106 GAPDH的患者临床获得完全缓解(CR).1例患者移植后近3个月时IgH拷贝数为4.5×103/106 GAPDH,4个月时临床复发.RQ-PCR检测8例干细胞采集物的肿瘤污染水平为3.68×102(0-1720)/106 GAPDH,外周血采集物中的肿瘤污染小于骨髓[75(0-890)/106 GAPDH vs 1.1×103(527-1720)/106 GAPDH,p＜0.05].采集物可用于RQ-PCR检测的8例患者无论肿瘤污染程度如何,临床均无复发.采集物中肿瘤污染的水平与初治及移植后1个月的IgH水平呈正相关(r值分别为0.810、0.708,p＜0.05).结论RQ PCR能够有效监测B细胞恶性肿瘤患者移植后IgH水平动态变化.移植后3个月内IgH拷贝数大于103/106 GAPDH可能是预测患者复发的标志.     

Episomal amplification of MYCN in a case of medulloblastoma

Gene amplification, in the form of double minutes (dmin) and/or homogeneously staining regions (hsr), is frequently associated with tumor development. A well-known example is neuroblastoma for which MYCN gene (v-myc myelocytomatosis viral-related oncogene) amplification has a relevant prognostic significance. A third cryptic form of amplification, cytogenetically invisible and composed of episomes, has been also described, but it is very rarely seen in primary tumors. In this paper, we report on MYCN amplification, in the form of episomes, in a case of medulloblastoma. Detailed fluorescence in situ hybridization and real-time quantitative polymerase chain reaction analyses revealed an amplified genomic segment of approximately 590 kb containing only the genes MYCN and N-cym (v-myc myelocytomatosis viral-related oncogene, neuroblastoma-derived opposite strand). To the best of our knowledge, this is the first report of a solid primary tumor showing MYCN amplification in the form of episomes.