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目的 用 1例有 3种免疫球蛋白分泌的多发性骨髓瘤 (MM)患者免疫球蛋白重链 (IgH)基因标志探讨MM究竟是由几个克隆组成。方法 患者血清蛋白电泳发现 3个单克隆免疫球蛋白峰 ;RT PCR采用IgHV 6个家族 (VH1～VH6)正向引物 ,通用J区反向引物 ,IgH恒区Cμ、Cδ、Cγ、Cα和Cε 5种特异性反向引物。采用不同的引物组合进行PCR。获得的免疫球蛋白重排基因经克隆后 ,进行测序分析。结果 患者用VH3家族引物和通用J区引物获得一条边缘清晰的浓染条带 ,表明患者骨髓瘤细胞是由IgVH3片段组成可变区。用IgVH3和恒区Cμ、Cγ、Cα特异性引物也可获得相应的PCR产物条带 ,但是不能用引物VH1、VH2、VH4、VH5和J以及VH3和Cδ、Cε获得 ,表明患者有IgM、IgG、IgA基因重排 ,因此外周血血清有相应免疫球蛋白产物。克隆用Cμ、Cγ、Cα引物获得的PCR产物并扩增 ,然后测序比较 ,3种产物除恒区片段不同外 ,其重链基因的VH J重排序列完全一致。表明该MM患者血清出现的 3种免疫球蛋白IgG、IgA、IgM的可变区是完全一致的。结论 尽管从蛋白角度分析该MM细胞似乎是寡克隆性的 ,但是基因序列分析的结果表明其恶性克隆仍然是单克隆性起源。
Objective To investigate the immunoglobulin heavy chain (IgH) gene signature of a patient with multiple myeloma (MM) with 3 kinds of immunoglobulin secretion and to investigate whether MM consists of several clones. Methods Three monoclonal immunoglobulin peaks were detected by serum protein electrophoresis. RT PCR was performed using IgHV 6 families (VH1-VH6) forward primers, universal J-region reverse primers, IgH constant regions Cμ, Cδ, Cγ, Cα and Cε. Five specific reverse primers. PCR was performed using different primer combinations. The obtained immunoglobulin rearrangement gene was cloned and sequenced. Results A patient’s VH3-family primers and universal J-region primers were used to obtain a borderline, densely stained band, indicating that the patient’s myeloma cells are composed of IgVH3 fragments that make up the variable region. The corresponding PCR product bands were also obtained with IgVH3 and constant region Cμ, Cγ, and Cα specific primers but could not be obtained with the primers VH1, VH2, VH4, VH5 and J and VH3 and Cδ, Cε, indicating that the patient has IgM, IgG , IgA gene rearrangement, so peripheral blood serum corresponding immunoglobulin products. The PCR products obtained with primers Cμ, Cγ, and Cα were cloned and amplified. After sequencing and comparison, the VHJ rearrangement sequences of the heavy chain genes of the three products were identical except that the constant region fragments were different. The variable regions of the three immunoglobulin IgG, IgA, and IgM appearing in the serum of the MM patient are completely identical. Conclusion Although the MM cell appears to be oligoclonal from the point of view of the protein, the results of gene sequence analysis indicate that its malignant clone is still of monoclonal origin.