降钙素基因逆转录巢式PCR及其临床应用价值的研究  

A STUDY OF RT-NESTED PCR AMPLIFICATION ON THE CALCITONIN GENE AND ITS CLINICAL VALUE

作　　者：秦燕[1] 陆德炎[1] 姚登福[2] 陆建新[2] 丁润生[1] 董志珍[1] 

机构地区：[1]南通医学院附属医院血液研究室,南通226001 [2]南通医学院附属医院,南通226001

出　　处：《南通医学院学报》1999年第4期392-399,共8页ACTA Academiae Medicinae Nantong

摘　　要：目的 :建立逆转录巢式 ( RT-nested) PCR方法扩增降钙素 ( CT)基因 ,探讨急性白血病诱导分化前后 CT基因甲基化模式以及在微小残留病灶 ( MRD)诊断中的临床价值。方法提取骨髓单个核细胞 ( BMMC)中的总 RNA,用逆转录酶合成 c DNA,再以巢式 PCR扩增含有 M位点的 CT基因片断 ,并以 Hpa 酶切 PCR扩增终产物 ,分析 CT基因的甲基化模式。结果 :( 1)所建立的 RT-nested PCR法检测 CT基因异常甲基化 ,其灵敏度为 1/ 2 0万 ,比 DNA直接 PCR法高 2 0 0倍 ;( 2 )观察到 CT基因高甲基化状态随着白血病细胞形态和功能上的成熟而部分改变。结论 :灵敏的 RT-nested PCR法分析 CT基因 ,可作为Objective: To investigate the methylation patterns of calcitonin (CT)gene of acute leukemia in the induction differentiation process and the clinical value of its detection on minimal residual disease by RT-nested PCR method.Methods: Extracting RNA from bone marrow mononucleated cells,cDNA synthesised with retroanscriptase,followed by amplifying CT gene fragment including M 4 site by nested PCR,digested the PCR products with HpaⅡ endonuclease,and then analysed the methylation patterns of CT gene. Results: (1)The sensitivity of the new assay,RT-nested PCR,on the CT gene hypermethylation is 1/200 thousand,and it is 200 times than DNA direct PCR method. (2)The CT gene hypermethylation changed partly along with induction differentiation of leukemia cells in vitro.Conclusions:The sensitive assay on the CT gene abnormalities in methylation may serve as a universal marker for detection of minimal residual disease during complete remission.

关 键 词：降钙素基因 白血病 微小残留病灶 RT-PCR 

分 类 号：R733.704[医药卫生—肿瘤]