石蜡淋巴瘤组织中IgH FR3区基因重排引物分析

Analysis of immunoglobulin heavy chain gene rearrangement by the frame region 3 primers and their application in paraffin-embedded lymphoma tissues

目的利用生物信息学方法分析免疫球蛋白重链(IgH)框架区(FR3)基因重排引物并探讨其在石蜡包埋非霍奇金淋巴瘤(NHL)组织中应用价值。方法通过ClustalW软件比较44条有效的IgH可变区和6条J区的基因片段,选取IgHFR3区3对(P1,P2,P3)基因重排引物,其中,P2为改进的引物。另选一对TCRγ引物作为T细胞淋巴瘤重排引物,通过PCR扩增,检测经形态学及免疫组织化学确诊的144例石蜡包埋组织标本,包括113例B细胞淋巴瘤、24例T细胞淋巴瘤和7例淋巴结反应性增生组织。以DG75淋巴瘤细胞系DNA作为对照组。结果引物对P1、P2、P3在B细胞淋巴瘤检出阳性率分别为71.7%(81/113),82.3%(93/113)和76.1%(86/113),三者检出率差异无统计学意义;在T细胞淋巴瘤检出率分别12.5%(3/24)、12.5%(3/24)、16.7%(4/24)。将P1和P2引物组合分析,B细胞淋巴瘤阳性检出率可以达到92.3%。以上重排引物在7例反应性淋巴结中均未检出。结论3对IgHFR3区中,新改进的P2引物在B细胞淋巴瘤中的检出率最高(82.3%)。2对IgHFR3区引物联合检测可明显提高石蜡组织中B细胞淋巴瘤的检出率。

Purpose To analyze immunoglobulin heavy chain （IgH） rearrangement by frame region 3 （FR3） primers and to explore their application in paraffin-embedded lymphoma tissues. Methods Three pairs of primers from IgH FR3 region （ P1, P2 and P3 ） were chosen with comparison of 44 IgH variable and 6 joined region gene fragments by the Clustal W software,of which the P2 primer was modified. 144 cases of histopathologically confirmed lymphoproliferative samples included 113 cases of B cell lymphoma, 24 cases of T cell lymphoma, and 7 cases of reactive proliferative lymph nodes. The DNA from DG75 and Jurkat cell lines were employed as positive controls of B and T cell lymphoma, respectivdy. Results The positive rates of IgH FR3 region primers （ P1, P2 and P3） were 71.7% （81/113）, 82.3% （93/113）, and 76. 1% （86/113） in 113 cases of B cell lymphoma, and 12. 5% （3/24）, 12. 5% （3/24）, and 16. 7% （4/24） in 24 cases of T cell lymphoma, respectively. There was no significantly statistical difference among three IgH FR3 primers in detection of 113 cases of B cell lymphomas. The positive rate could be increased to 92. 3% by combined use of the P1 and P2 primers. There were no positive cases among 7 cases of proliferative lymphoid tissues. Conclusion Among 3 IgH FR3 region primers, newly modified P2 primer has a high detection rate in the gene rearrangement of paraffin-embedded lymphoma tissues. The detection rate could be increased by combining the two IgH FR3 primers in the IgH gene rearrangement analysis.