WT1基因定量分析在儿童急性白血病MRD检测中的研究

WTl基因定量分析在儿童急性白血病MRD检测中的研究中文摘要目的：采用实时荧光定量PCR(RQPCR)方法检测WTl基因在急性白血病患JLPb周血和骨髓中的表达，探讨WTl基因在检测微小残留病(minimalresidual disease，MRD)中的价值。方法：采用实时荧光定量PCR方法，检测23例初治急性白血病患儿、80例次完全缓解患儿外周血及骨髓中WTl基因的表达水平，以10例非肿瘤患儿为正常对照；流式细胞术检测MRD，并同时观察白血病患儿疾病状态。23例初治急性白血病患儿包括16例急性淋巴细胞白血病(ALL)及7例急性粒细胞白血病(AML)，其中初治ALL组于治疗前、治疗第1 5天、33天及3个月，初治AML组于治疗前、治疗第15天、1个月、3个月分别采集外周血和骨髓标本，检测WTl表达。80例次完全缓解组患儿(ALL56例次，AML24例次)于治疗第3个月、6个月及12个月分别采集外周血和骨髓检测WTl表达阳性率。结果：1、正常对照组儿童外周血及骨髓WTl表达阳性率均为0，初治白血病组患,SlA,b周血及骨髓WTl表达阳性率均为652，初治白血病组外lIII I I III II I I II II t i llIY2338662周血及骨髓WTl的表达均明显高于正常对照组(p=O001)。ALL和AML外周血WTl表达阳性率比较，骨髓WTl表达阳性率比较，差异均无统计学意义(p-0533)。完全缓解患儿治疗第3个月、6个月外周血及骨髓WTl表达阳性率明显高于第12个月(p均O05)。2、初治ALL患儿治疗前、治疗第1 5天、33天及3个月外周血WTl表达量分别为053+014、024士013、01 8+005、015a：01 1，各组间差异具有统计学意义(F=2468，PO001)；治疗第15天、33天及3个月WTl表达量明显低于治疗前(p均005)。初治ALL患儿治疗前、15天、33天及3个月骨髓WTl表达量分别为072-4-014、016-4-006、O12士O02、O12+005，各组间差异具有统计学意义(F=13527，PO001)，治疗第15天、33天及3个月WTl表达量明显低于治疗前(p均O05)。所有初治ALL患儿皆于33天达完全缓解。3、初治AML患儿治疗前、15天、1个月及3个月外周血WTl表达量分别为070-a：007、020a：006、O18土005、015-4-006，各组间差异具有统计学意义(F=13929，PO001)，治疗第15天、33天及3个月WTl表达量明显低于治疗前(p均O05)。初治AML患儿治疗前、治疗第15天、1个月及3个月患儿骨髓WTl表达量分别为O87：1：004、024+005、021：士-005、O18士O10，各组间差异具有统计学意义(F=24129，P0001)，治疗第15天、33天及3个月WTl表达量明显低于治疗前(p均O05)。全部初治AML患儿皆于1-2个月内获得完全缓解。4、初治ALL患JLJ，l-周血WTl表达量与MRD成正相关， (FO999，PO001)；初治ALL患儿骨髓WTl表达量与MRD成正相关(r=O997，Po001)。5、3例复发白血病患儿复发前3个月WTl水平上升。结论：1、实时荧光定量PCR方法检测白血病患儿WTl表达简便易行、准确性高、特异性好。2WTl基因在非肿瘤儿童外周血及骨髓均不表达，而在急性白血病治疗前高表达。3初治急性白血病患儿诱导缓解化疗后WTl表达水平明显低于初治时。4WTl阳性率随着治疗时间的延长明显下降。5ALL患)hil-周血及骨髓中WTl表达量与流式细胞仪检测的MRD呈正相关。对急性白血病患Jl,J,b周血及骨髓中WTl基因表达进行定量分析，可用于MRD的监测。硕士研究生尹栓(儿科学专业)指导教师 李学荣副教授【关键词】WTl；急性白血病；微小残留病；实时荧光定量PCRStudy of quantification of WTl for monitoring minimal residual diseasein childhood acute leukemiaAbstractObjective：To detect the expression of WTl gene in peripheral blood(PB)and bone marrow(BM)by using real time quantitative PCR(RQ-PCR)，toexplore its value for monitoring minimal residual disease in children withacute leukemiaMethods：WTl gene was detected with RQPCR in PB and BM from 23denovo AL children and 80 children with complete remission(CR)，1 0 casesof nonmalignant disease were used as control groupFlow Cytometry(FCM)was used to determine MRD and observe the disease process of AL children23 cases were preliminary diagnosis patients，including 1 6 acutelymphoblastic leukemia(ALL)and 7 acute myeloid leukemia(AML)patientsBM and PB from ALL children before treatment and on the 1 5thday、3 3th day and third month were collectedBM and PB from AMLchildren before treatment and on the 1 5th day、first month and third monthwere collectedComplete remission group including 56 ALL and 24 AMLcases，BM and PB on the third month、sixth month and twelfth month werecollectedResults：(1)The positive expression rate of WTl were 0 and 652respectively in PB and BM from control group and denovo AL childrenThepositive expression rate of WTl gene in childhood acute leukemia was higherthan normal controls(p-OOO 1)There was no statistic difference PB cellsWTl positive expression rate betweet ALL and AML(p=O533)There wasno statistic difference BM cells WT l positive expression rate betweet ALLand AML(p=O533)The positive expression rate of WTl gene on the thirdmonth、sixth month was higher than twelfth month in complete remissiongroup(pO05)(2)WTl gene expression showed 053+01 4、024+01 3、O1 8士005、O1 54-01 1 in PB cells before treatment and on the 1 5th day、33thday and third month respectively in denovo ALL children，significantdifference was found(F=2468，p000 1)The expression of WTI gene levelsbefore treatment was higher than the 1 5th day、33th day and third month inPB(pO05)WTl gene expression showed0724-01 4、0164-006、O12士O02、O12士O05 in BM cells before treatment and on the1 5th day、33th day and third month respectively in denovo ALLchildren，significant difference was found(F=1 3 527，p000 1)The expressionof WTl gene levels before treatment was higher than the 1 5th day、3 3th dayand third month in BM(pO05)1 6 denovo ALL childrenwere complete remission on the 33th day(3)WTl gene expression showed070+007、020+006、O1 8+005、O1 5+006 in PB cells before treatment andon the 1 5th day、first month and third month respectively in denovo AMLchildren，significant difference was found(F=1 3929，p000 1)Theexpression of WTl gene levels before treatment was higher than the 1 5thday、first month and third month in PB(p005)WTlgene expression showed087+004、0241005、021+005、O18+010 in BMcells before treatment and on the 1 5th day、first month and third monthrespectively in denovo AML children，significant difference wasfound(F=24 129，p000 1)The expression of WTl gene levels beforetreatment was higher than the 1 5th day、first month and third month inBM(pO05)7 denovo AML children werecomplete remission on the first month(4)MRD was positively related toWT 1 gene levels in PB(r=O999，PO00 1)MRD was positively related toWTl gene levels in BM(r=O997，p0001)(5)When the patients wererelapsed，the WTl gene levels increaseConelusions：(1)The real time quantitmive PCR assay has good sensitivity，repeatability and specificity(2)There was no expression in children withoutmalignant disease，but the expression was high before treatment in denovoAL children(3)The levels of WT 1 gene in complete remission group werelower than in denovo AL children(4)With the development of treatment，thepositive expression rate of WTl was declined significantly(5)MRD waspositively related to WT 1 gene levels in denovo ALL childrenMonitoringWT 1 with RQPCR can act as a method to detect minimal residual disease inchildren with acute leukemiaPostgraduate student：Yin Shuan(peadiatries)Directed by Profi Li XuerongKey words：WTl；Acute leukemia；Minimal residue disease；Real time quantitativePCR目录弓I言1第1章材料与方法211材料2111研究对象2112实验试剂2113实验仪器312 WTl定量分析方法4121标本采集与保存4122总RNA的提取4123 RNA浓度检测及鉴定”