顾伟英&nbsp; 陈子兴&nbsp; 曹祥山&nbsp; 胡绍燕&nbsp; 朱江&nbsp; 王志林&nbsp; 严峰&nbsp; 王玮&nbsp; 岑建农&nbsp; 沈慧玲&nbsp; 钱军<BR>[摘要]&nbsp; 目的&nbsp; 探讨急性白血病患者骨髓中WT1的表达水平。&nbsp; 方法&nbsp; 建立实时定量RT-PCR方法，采用LightCycler PCR仪检测了108例急性白血病患者和23例非白血病患者骨髓中WT1及内参GAPDH的表达水平，以WT1<SUB>N</SUB> =（WT1拷贝数/GAPDH拷贝数）×10<SUP>4</SUP> 计算WT1表达水平。 结果&nbsp; 初诊与复发AL患者骨髓中WT1N的中位表达水平分别为75.10和89.56，明显高于CR组和对照组（分别为2.07和1.51），对照组与CR组之间及初诊与复发组之间无统计学差异；70例初诊急性白血病中ALL、M<SUB>1</SUB>、M<SUB>2</SUB>、M<SUB>3</SUB>亚型WT1N 表达水平明显高于M<SUB>5</SUB>，即粒系表达明显高于单核系，且WT1表达水平与外周血WBC计数、骨髓原始细胞比例、MDR<SUB>1</SUB>无相关性，但与染色体核型有关。对其中2例患者动态检测WT1可提示白血病复发。 结论&nbsp; WT1在急性白血病患者骨髓中高表达，可作为白血病疗效考核及监测残留病灶的指标。<BR>[关键词]&nbsp; 白血病，急性； 基因，WT1； 逆转录聚合酶链反应<BR>Detection of WT1 expression in bone marrow of acute leukemia with real-time quantitative RT-PCR&nbsp; GU Weiying, CHEN Zixing, CAO Xiangshan, HU Shaoyan.The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou&nbsp; 215006, China<BR>[Abstract]&nbsp; Objective&nbsp; To investigate wilms’ tumor gene (WT1)&nbsp; expression levels in bone marrow(BM) of acute leukemia patients(Als). Methods&nbsp; Real-time quantitative retroverse polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in BM of 108 ALs and 23 non-leukemias by LightCycler. Normalized WT1 expression level (WT1N ) was determined as a ratio between WT1 and GAPDH times 10<SUP>4</SUP>. Results&nbsp; The Median expression levels of WT1N in 70 newly diagnosed ALs&nbsp; and 11 relapsed Als were statistically higher than those of 23 ALs with complete remission（CR）&nbsp; and&nbsp; 23 non-leukemic&nbsp; controls(75.10&nbsp; and&nbsp; 89.56&nbsp; vs 2.07&nbsp; and&nbsp; 1.51&nbsp; ). No statistic differences were found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the&nbsp; 70 newly diagnosed Als ,Median WT1N expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias （M<SUB>3</SUB>），but there were no statistic differences among the M<SUB>1 </SUB>, M<SUB>2 </SUB>, M<SUB>3</SUB> and&nbsp; ALL&nbsp; subtypes. Furthermore the&nbsp; WT1N levels&nbsp; were not correlated to&nbsp; WBC counts&nbsp; of peripheral blood, blast ratio of BM and multidrug resistant gene（MDR1） expression at presentation, but correlated to chromosome types.Dynamics of WT1N levels of 2 patients during treatment showed that WT1 expression levels could prognose relapse . Conclusion&nbsp; WT1 expression levels in ALs were strikingly higher than in non-leukemias who were undectable or lower expression. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.<BR>[key words]&nbsp; leukemia,acute; WT1; RT-PCR; real-time quantitative
WT1（Wilms’ tumor gene,WT1）定位于11p13，是最早发现与Wilms’肿瘤发生、发展有关的基因。 WT1主要在胚胎发生过程中表达，对泌尿生殖系统的发育起重要作用，在成人，WT1仅在肾脏、卵巢、子宫内膜、睾丸、脾脏、乳腺及正常造血祖细胞微量表达^[1]。研究发现几乎所有的白血病原始细胞（无论系别）都持续过表达WT1，与其在极少数生理性造血干细胞（CD34 ）中短暂低表达形成鲜明对比，定性和半定量RT-PCR法在区别单个核细胞WT1是生理性还是病理性表达存在缺陷和限制，WT1作为“泛白血病标志”（panleukemic marker）在急性白血病中的表达及预后意义是一个有争论的问题[2]，为进一步准确检测，我们采用实时定量RT-PCR技术检测了108例急性白血病患者骨髓中WT1的表达水平，报道如下：
对象与方法
1 研究对象 所有的白血病患者均来自苏州大学附一院及附三院血液科2002年3月~2003年10月门诊或住院治疗的患者，男62例，女46例，年龄6岁~72岁之间，中位年龄36 .5岁。白血病的诊断均经临床、血象、骨髓象及组织化学染色、免疫分型、染色体检查确诊，部份患者行融合基因检测，符合白血病MIC分型。其中AML 95例（M0 2例，M₁ 20例，M₂ 38 例，M₃ 18例， M₄ 7例， M₅ 10例 ），ALL 13例。对照组23例，为同期就诊非白血病患者（过敏性紫癜，缺铁性贫血 ，免疫性血小板减少性紫癜，巨幼细胞性贫血，淋巴瘤）及健康供者CD34细胞。以WT1高表达的K562细胞作为阳性对照。
2 主要试剂 TRIzol总RNA提取液购自Gibco BRL公司，逆转录酶M-MLV购自Promega公司，Taq DNA聚合酶购自上海申友公司。
3 骨髓单个核细胞分离：常规抽取患者骨髓液2~5ml，肝素抗凝，用淋巴细胞分离液（Ficoll分离液）分离单个核细胞，计数（5~10）×10⁶细胞加TRIzol总RNA提取液1ml，混匀后-20℃冻存（时间小于2月）用于RNA制备。
4 RNA提取及定量：采用TRIzol一步法提取骨髓单个核细胞总RNA，紫外分光光度仪测定OD