骨髓瘤细胞免疫球蛋白重链基因可变区的研究

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

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。

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 -