一步及半巢式PCR检测石蜡包埋B细胞淋巴瘤组织克隆性IgH重排的比较

DETECTION OF IMMUNOGLOBULIN HEAVY-CHAIN GENE REARRANGEMENT OF FORMALIN-FIXED AND PARAFFIN-EMBEDDED B CELL LYMPHOMA TISSUE BY SINGLE-STEP AND SEMI-NESTED POLYMERASE CHAIN REACTION

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 set up a simple and effective method to detect clonal immunoglobulin heavy chain gene rearrangement of formalin fixed and paraffin embedded B cell lymphoma tissue.Methods:Genomic DNA was extracted from formalin fixed paraffin embedded tissue of 36 B cell lymphomas and 10 chronic tonsillitis by phenol chloroform and/or boiling method.Then the third complementarity determining region of immunoglobulin heavy chain(IgH CDR 3) gene rearrangement was analyzed by single step and semi nested polymerase chain reaction(SnPCR),polyacrylamide gel electrophoresis and silver staining.Results:In the group of B cell lymphomas,clonal IgH CDR 3 rearrangement was detected in 26 cases(76 5%) and in 9 cases(26 5%) by SnPCR and single step PCR,respectively,when DNA of 34 cases was extracted by phenol chloroform method;and in 18 cases(66 7%) and 2 cases(7 4%) by SnPCR and single step procedure,respectively,when DNA of 27 cases was extracted from tissues boiling method.SnPCR detected more clonal IgH gene rearrangement than single step PCR did( P <0 05).In 25 cases,DNA was extracted using both techniques,the clonal IgH CDR 3 rearrangement was detected in 76% and 68% respectively( P >0 05) of them by SnPCR.Conclusion:Monoclonality of B cell lymphomas can be effectively evaluated by PCR equally well from formalin fixed,paraffin embedded tissues DNA extracted by boiling and phenol chloroform method.The former is more simple,lower in cost and less poisonous.SnPCR identified more clonal IgH CDR 3 gene rearrangement than single step PCR did in formalin fixed,paraffin embedded tissues.