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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 MRD监测的通用基因标志
Objective : To establish a reverse transcription RT-nested PCR method for the amplification of calcitonin (CT) gene and to explore the clinical value of CT gene methylation patterns before and after differentiation of acute leukemia in the diagnosis of minimal residual disease (MRD). Methods The total RNA in bone marrow mononuclear cells (BMMC) was extracted, and cDNA was synthesized by reverse transcriptase. Then the CT gene fragment containing M sites was amplified by nested PCR, and the final product was amplified by Hpa enzyme digestion and analyzed. The methylation pattern of the CT gene. Results: (1) RT-nested PCR was used to detect abnormal methylation of CT gene with a sensitivity of 1/200 000, 200 times higher than that of direct DNA PCR; (2) Hypermethylation of CT gene was observed. The state changes partially with the maturity and function of leukemic cells. Conclusion : Sensitive RT-nested PCR analysis of CT gene can be used as a universal gene marker for MRD monitoring