多重实时荧光定量PCR同时检测急性白血病患者WT1和MDR1基因表达水平方法的建立
다중실시형광정량PCR동시검측급성백혈병환자WT1화MDR1기인표체수평방법적건립
Simultaneous detection of WT1 and MDR1 gene expression in acute leukemia patients by a multiplex real time quantitative PCR method
  • 万方数据
    中华检验医学杂志 중화검험의학잡지
    CHINESE JOURNAL OF LABORATORY MEDICINE
    2011年 1期 15-19 ,共5页

    徐兵%宋小燕%杨柳%许文娟%黄芬%郭绪涛%周淑芸

    서병%송소연%양류%허문연%황분%곽서도%주숙예

    白血病%WT1蛋白质类%P糖蛋白%聚合酶链反应 白血病%WT1蛋白質類%P糖蛋白%聚閤酶鏈反應
    백혈병%WT1단백질류%P당단백%취합매련반응
    Leukemia%WT1 proteins%P-Glycoprotein%Polymerase chain reaction
    目的 构建可同时检测AL患者WT1和MDR1基因表达水平的多重实时荧光定量PCR方法.方法 提取K562细胞的总RNA,并逆转录扩增WT1和MDR1的cDNA,通过Bam H Ⅰ及BglⅡ酶切后连接成WT1和MDR1重组片段,对WT1和MDR1重组片段进行PCR扩增,PCR产物纯化后与pMD18载体进行连接,转化宿主菌DH-5α,构建载有WT1和MDR1基因的标准品重组质粒,用限制性核酸内切酶法和PCR法鉴定质粒.用FAM荧光标记MDR1探针、VIC荧光标记WT1探针,建立能在1个试管中同时检测WT1和MDR1基因表达水平的多重实时荧光定量PCR反应体系.应用该体系检测47例AL患者及32例对照者WT1和MDR1基因表达水平,比较两组之间WT1和MDR1基因表达水平的差异,并随访7例AL患者不同病程中WT1和MDR1基因表达水平的变化与临床预后的关系.结果 经EcoR1酶切及PCR法证实WT1和MDR1基因重组质粒已成功构建.所建立多重实时荧光定量PCR方法的灵敏度为102拷贝/μl;所建立标准曲线的R2分别为0.999和0.998.AL患者WT1和MDR1基因的表达水平中位数分别为37 000(163～6 370 000)拷贝/μg RNA和76 200(179～18 000 000)拷贝/μg RNA,显著高于对照组的258(0～643)拷贝/μg RNA和333(0～779)拷贝/μg RNA,差异有统计学意义(Z=6.755、6.736,P＜0.01).对应用相同的诱导和巩固化疗方案治疗的7例AL患者进行随访,3例持续缓解患者中,WT1和MDR1的表达水平在初治时分别为2 170和86 900、1 130和5 860、1 170和586拷贝/μg RNA,治疗后WT1和MDR1的表达水平明显下降,分别为370和560、138和980、150和690拷贝/μg RNA,此3例患者获长期完全缓解.在3例完全缓解后复发患者中,WT1和MDR1表达水平在初治时分别为1 600和11800、24 800和968、48 200和1 100 000拷贝/μg RNA,在化疗获得完全缓解后均降低,但在随后治疗过程中,WT1、MDR1表达量又逐渐升高,分别为20 314和25 660、184 364和31 530、15 680和878 000拷贝/μg RNA,此3例患者最终均出现临床复发.另外1例未缓解患者初治时WT1和MDR1表达水平分别为81 600和1 200 000拷贝/μg RNA,化疗后WT1、MDR1表达水平不仅未下降,反而有所上升,分别为124 100和7 632 400拷贝/μg RNA,此例患者始终未获缓解.结论 本研究成功构建了可同时检测WT1和MDR1基因表达水平的多重实时荧光定量PCR方法.同时检测AL患者WT1和MDR1基因表达水平的变化可能有助于判断预后.
    목적 구건가동시검측AL환자WT1화MDR1기인표체수평적다중실시형광정량PCR방법.방법 제취K562세포적총RNA,병역전록확증WT1화MDR1적cDNA,통과Bam H Ⅰ급BglⅡ매절후련접성WT1화MDR1중조편단,대WT1화MDR1중조편단진행PCR확증,PCR산물순화후여pMD18재체진행련접,전화숙주균DH-5α,구건재유WT1화MDR1기인적표준품중조질립,용한제성핵산내절매법화PCR법감정질립.용FAM형광표기MDR1탐침、VIC형광표기WT1탐침,건립능재1개시관중동시검측WT1화MDR1기인표체수평적다중실시형광정량PCR반응체계.응용해체계검측47례AL환자급32례대조자WT1화MDR1기인표체수평,비교량조지간WT1화MDR1기인표체수평적차이,병수방7례AL환자불동병정중WT1화MDR1기인표체수평적변화여림상예후적관계.결과 경EcoR1매절급PCR법증실WT1화MDR1기인중조질립이성공구건.소건립다중실시형광정량PCR방법적령민도위102고패/μl;소건립표준곡선적R2분별위0.999화0.998.AL환자WT1화MDR1기인적표체수평중위수분별위37 000(163～6 370 000)고패/μg RNA화76 200(179～18 000 000)고패/μg RNA,현저고우대조조적258(0～643)고패/μg RNA화333(0～779)고패/μg RNA,차이유통계학의의(Z=6.755、6.736,P＜0.01).대응용상동적유도화공고화료방안치료적7례AL환자진행수방,3례지속완해환자중,WT1화MDR1적표체수평재초치시분별위2 170화86 900、1 130화5 860、1 170화586고패/μg RNA,치료후WT1화MDR1적표체수평명현하강,분별위370화560、138화980、150화690고패/μg RNA,차3례환자획장기완전완해.재3례완전완해후복발환자중,WT1화MDR1표체수평재초치시분별위1 600화11800、24 800화968、48 200화1 100 000고패/μg RNA,재화료획득완전완해후균강저,단재수후치료과정중,WT1、MDR1표체량우축점승고,분별위20 314화25 660、184 364화31 530、15 680화878 000고패/μg RNA,차3례환자최종균출현림상복발.령외1례미완해환자초치시WT1화MDR1표체수평분별위81 600화1 200 000고패/μg RNA,화료후WT1、MDR1표체수평불부미하강,반이유소상승,분별위124 100화7 632 400고패/μg RNA,차례환자시종미획완해.결론 본연구성공구건료가동시검측WT1화MDR1기인표체수평적다중실시형광정량PCR방법.동시검측AL환자WT1화MDR1기인표체수평적변화가능유조우판단예후.
    Objective To set up a multiplex real time quantitative PCR method to detect the expression of WT1 and MDR1 gene simultaneously in acute leukemia patient. Methods Total RNA was extracted from k562 cell line and was reverse transcribed to cDNA by the outer primers of WT1 and MDR1 respectively. The cDNA of WT1 and MDR1 were purified and digested by Bam H Ⅰ and Bgl Ⅱ , and then the two fragments were ligated to form the recombinant fragment WT1 + MDR1. The outer forward primer of WT1 and outer reverse primer of MDR1 were used to amplify the recombinant fragment WT1 + MDR1. The PCR product was purified and cloned into pMD18-T vector, and then transferred into E. coli DH-5α. A new kind of WT1-MDRl-contained standard plasmid was obtained from the positive colony. The recombinant plasmid was verified by digestion with restriction enzyme and PCR amplification. A multiplex real time quantitative PCR method was set up with FAM-labeled MDR1 probe and VIC-labeled WT1 probe in one reaction tube. The WT1 and MDR1 gene expression was detected in forty-seven AL patients and thirty-two controls by this method. Seven patients were followed-up to elucidate the relationship between the gene expression levels and clinical prognosis. Results The recombinant plasmid was confirmed by EcoR1 digestion and PCR amplification. The multiplex real time quantitative PCR technique could reach the sensitivity of WT1 and MDR1 gene up to 102 copy/μl. The standard curve slopes were 0. 999 and 0. 998. The WT1 [ 37 000( 163-6 370 000 )copies/μg RNA ] and MDR1 [ 76 200( 179-18 000 000 )copies/μg RNA ]expression levels of AL patients were significantly higher as compared to the controls [ 258( 0-643 ) copies/μg RNA and 333( 0-779 )copies/μg RNA ]( Z= 6. 755,6. 736, P ＜ 0. 01 ). Following up seven patients with similar regimen of chemotherapy, the WT1 and MDR1 expression correlated to the clinical course. Three AL patients with WT1 and MDR1 expression levels (2 170 and 86 900, 1 130 and 5 860, 1 170 and 586 copies/μg RNA )significantly decreased after chemotherapy and kept in the low range ( 370 and 560,138 and 980, 150 and 690 copies/μg RNA ), and had a favorable outcome. Three AL patients with WT1 and MDR1 expression levels ( 1 600 and 11 800, 24 800 and 968, 48 200 and 1 100 000 copies/μg RNA )decreased after initial chemotherapy, but increased significantly afterwards (20 314 and 25 660,184 364 and 31 530, 15 680 and 878 000 copies/μg RNA ),and suffered clinical relapse. One patient with high WT1 and MDR1 expression levels ( from 81 600 and 1 200 000 copies/μg RNA to 124 100 and 7 632 400 copies/μg RNA )showed the persistence of disease. Conclusions A multiplex real time quantitative PCR method to detect WT1 and MDR1 gene simultaneously is constructed successfully. The expression of WT1 and MDR1 may provide useful information for AL patients prognosis.
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