摘要
目的建立一个用于测定多药耐药基因(mdr_1)表达水平、反映临床耐药情况的逆转录-聚合酶链反应(RT-PCR)半定量技术。方法根据处于指数增长期的 PCR 产物量可反映最初摸板量多少的原理,以白血病为疾病模型,通过对最佳模板量及最佳循坏数的分析、参比对照的设定等,建立了^(35)S-dATP(~35硫-脱氧三磷酸腺苷)掺入的 RT-PCR 法,并以此法对19例临床白血病标本 mdr_1mR-NA(信使核糖核酸)表达水平进行了检测。结果 19例临床标本检测发现,6例化疗耐药或复发病例中,5例有高水平的 mdr_1mRNA 表达(mdr_1/β-actin:0.68～0.76)(β-actin:β肌动蛋白)。11例化疗完全缓解或部分缓解病例中,除2例有低水平 mdr_1mRNA 表达(mdr_1/β-actin:0.33、0.40)外,其余9例均为阴性。mdr_1mRNA 表达水平和临床化疗结果明显相关(P=0.017)。结论所建立的方法基本上可反映白血病患者化疗耐药情况,指导临床化疗。
Objective To develop a sensitive,specific and reproducible quantitation technique for e- valuating mdr1 gene expression and use as a guide for the chemotherapy.Methods Based on the princi- ple that the quantity of PCR product is a reflection of the quantity of template at the exponential increas- ing stage.A reverse transcription-polymerase chain reaction(RT-PCR)quantitation technique of mdr1 gene expression was developed through the determination of the optimal template quantity and the opti- mal cycle number,the setting of internal control and ~35S-dATP incorportion.Busing the present tech- nique,19 leukemia patients were examined.Results In 19 cases,out of 6 cases with relapse or no re- sponse to chemotherapy,5 expressed high level of mdr1 mRNA(mdr1/β-actin:0.68～0.76).Among 11 patients with complete remission or partial remission,9 cases were mdr1 expression negative and 2 low level of mdr1 mRNA expression(mdr1/β-actin:0.33,0.40).A good correlationship was noted be- tween mdr1 gene expression and the clinical apperance of the patients statistically.Conclusions The preliminary results suggest that this tchnique might be helpful in clinical treatment of leukaemia.
