骨髓瘤细胞免疫球蛋白重链基因可变区的研究

为分析多发性骨髓瘤 (multiplemyeloma,MM )细胞对免疫球蛋白重链基因可变区 (VH)基因家族的取用 ;根据VH 基因突变特点 ,揭示MM细胞的起源。以重链基因可变区 (VH1 VH6 )基因家族特异性引物 ,用PCR法扩增骨髓瘤细胞系CZ 1细胞和 98例MM患者外周血单个核细胞VH 基因片段 ,纯化后的PCR产物和pMD18 T载体连接并转化JM10 9细菌 ,经克隆鉴定后 ,目的DNA片段用末端双脱氧法测定DNA序列 ,和其对应的胚系基因序列比较。结果表明MM细胞对各VH 基因家族的取用顺序为VH3>VH1>VH4 >VH2 >VH5 >VH6 ;MM细胞VH 基因互补决定区 (CDR )氨基酸替换性突变 (R突变 ) /氨基酸静寂性突变 (S突变 )等于 9 6 7,而骨架区 (FR )R/S等于 0 87,而且随着疾病的进展 ,IgVH 基因并不发生进一步的突变。结论是MM前体细胞在进行VDJ基因重排时 ,对VH 基因家族的取用和基因家族相对大小有关 ;MM细胞可能起源于已经发生抗原选择和体细胞突变的B记忆细胞或前浆细胞。

The Study on the Variable Region of Immunoglobulin Heavy Chain Gene in Multiple Myeloma

To analysis the usage of variable region of heavy chain genes (V H) from the available repert oire of the multiple myeloma (MM) cell and to elucidat e the origin of the MM progenitor cell by analysis the charactertics of the gene mutation in V H, the DNA of the MM cell line CZ-1 cell and the peripheral blood mononucular cells of 98 MM patients were amplified by PCR technology using unique primers to variable region 1 to variable region 6 (V H1-V H6) The purified PCR products were inserted into pMD18 -T vector ,then transfected in JM109/ bacteria The expected DNA fragments wer e sequenced by dideoxy chain termination method,then compared with the correspo nding germ line gene sequences.The results showed that the turn of the usage of V H genes from the available repertoire in multiple myeloma was V H3>V H1>V H4>V H2>V H5>V H6 ;The amino replacement mutation (R)/ The amino silence mutation (S) ratio in complementary determined regions ( CDR) was 9 67,the R/S ratio in framework regions (FR) was 0 87,a further common feature was the lack of intraclonal variation in V H gene sequence from plate phase to advanced phase. It was concluded that the origin of the MM p rogenitor cell was B cell or pre-plasma cell which had experienced antigen sele ction and somatic mutation;the usage of V H genes from the available repertoir e in multiple myeloma appeared to reflect no striking bias, with predominance of the largest V H3 family -