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

Simultaneous detection of WT1 and MDR1 gene expression in acute leukemia patients by a multiplex real time quantitative PGR method

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.

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.