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目的 :建立一种简便有效的方法 ,来检测B细胞非霍奇金淋巴瘤 (B NHL)石蜡包埋组织的免疫球蛋白重链 (IgH)基因重排。 方法 :以IgH的第三互补决定簇区 (IgH CDR 3)为标志 ,用消化煮沸法和酚 氯仿法提取 36例B NHL及 1 0例扁桃体炎的石蜡包埋组织的基因组DNA ,比较两者进行一步及半巢式聚合酶链反应 (SnPCR) ,聚丙烯酰胺凝胶电泳、银染后检测克隆性IgH重排的结果。 结果 :B NHL组 ,34例经酚 氯仿法提取DNA ,其一步及SnPCR检测克隆性IgH CDR 3重排的阳性率分别为 2 6 5%和 76 5% (P <0 0 5) ;2 7例经消化煮沸法提取DNA ,其一步及SnPCR的阳性率分别为 7 4%和 66 7% (P <0 0 5) ;2 5例同时用上述 2种方法提取DNA ,SnPCR的阳性率分别为 76%和 68% (P >0 0 5)。对照组克隆性IgH重排阴性。结论 :消化煮沸法与酚 氯仿法提取的石蜡包埋组织的DNA均可用于IgH重排的检测 ,前者简便、经济、低毒 ,值得推荐。检测B NHL石蜡包埋组织的克隆性IgH重排 ,SnPCR优于一步PCR。
Objective: To establish a simple and effective method to detect immunoglobulin heavy chain (IgH) gene rearrangements in paraffin-embedded tissues of B-cell non-Hodgkin’s lymphoma (B NHL). Methods: The genomic DNA of paraffin-embedded tissues from 36 cases of B NHL and 10 cases of tonsillitis were extracted by digestion and boiling method and phenol-chloroform method with IgH CDR3 as the marker. One-step and semi-nested polymerase chain reaction (SnPCR), polyacrylamide gel electrophoresis, and silver staining were performed to detect clonal IgH rearrangements. Results: In the NHL group, DNA was extracted from 34 cases by phenol-chloroform method. The positive rates of cloned IgH CDR3 rearrangement in one step and SnPCR were 26.5% and 76.5%, respectively (P <0.05) DNA digestion and boiling were performed in one step and the positive rate of SnPCR was 74% and 667%, respectively (P <0.05). The positive rates of SnPCR in 25 cases with DNA extracted by the above two methods were 76% and 68% (P> 0.05). The control group clonal IgH rearrangement negative. Conclusion: The DNA of paraffin-embedded tissues extracted by digestion and boiling method and phenol-chloroform method can be used for the detection of IgH rearrangement. The former is simple, economical and low toxicity and is recommended. Clonal IgH rearrangement was detected in B-NHL paraffin-embedded tissue, and SnPCR was superior to one-step PCR.