荧光定量PCR分析弥漫大B细胞淋巴瘤免疫球蛋白重链基因重排表达  

The study of immunoglobulin heavy-chain gene rearrangement in diffuse large B-cell lymphoma using fluorescence quantitative PCR

作　　者：易基群[1,2] 林桐榆[1] 何友兼[1] 

机构地区：[1]华南肿瘤学国家重点实验室,中山大学附属肿瘤防治中心内科,广东广州510060 [2]广州市红十字会医院(暨南大学附属第四医院)肿瘤科

出　　处：《中国癌症杂志》2007年第8期619-623,共5页China Oncology

基　　金：广东省科技计划项目(NO:2003C30314)

摘　　要：背景与目的:大多数B细胞淋巴瘤患者综合治疗后可以达到完全缓解,但是一半以上的患者终究要复发。复发来源于体内残留的耐药淋巴瘤细胞,即微小残留病变。但临床上发现IgH基因重排阳性患者并非都出现复发或远处浸润。因此,推测阳性患者是否复发,可能与IgH基因重排的表达量有关。本研究探讨荧光染料标记的即时定量PCR方法检测弥漫大B细胞淋巴瘤免疫球蛋白重链基因(IgH)重排的可行性及临床意义。方法:44例DLBCL患者的57份新鲜骨髓标本用于检测IgH基因重排,Namalwa细胞系作阳性对照,U-937细胞系作阴性对照。β-actin作内参照,SYBRGreen荧光染料标记的实时荧光定量PCR方法分别检测IgH基因重排CDRIII。结果:分析融解曲线可以确定IgH基因重排产物的特异性。荧光定量PCR检测IgH基因重排的阳性率63.2%。IgH/β-actin阳性表达量在0.01～4131.69,中位数0.42。Ⅰ/Ⅱ期患者IgH基因重排表达量中位数为0,Ⅲ、Ⅳ期患者IgH基因重排表达量中位数为0.35,经统计学检验,两组患者之间差异有显著性(P=0.018)。LDH值高于正常组,IgH基因重排表达量为0.39,LDH值低于正常组,IgH基因重排表达量为0.01,经非参数检验,两组患者之间差异有显著性(P=0.046)。结论:荧光染料标记的定量PCR方法可用于弥漫大B细胞淋巴瘤的骨髓微小残留病变的检测。检测骨髓IgH基因重排,可以协助分期。Background and purpose： Majority of patients with B cell lymphoma often achieve complett clinical remissies after systemic treatment , but half of the patients ultimately relapse, The residual neoplastic cells, commonly called minimal residual disease＇ （MRD） , are thought to be the source of relapse. But not all of the patients whose resuhs of IgH rearrangement were poaitive had relapse or distant involvement, h was thought that lhe patients whose IgH reangement was positive relapsed or not may be assciated with the quantity of IgH rearrangement. The study tried to investigate the feasibility and clinical significance of detecting immunoglobulin heavy chain gene in DLBCI, hy SYBR Green RT-FQ-PCR. Methods： Fifty-seven bone marrow specimens from 44 patients diagnosed wilh DLBCL were used to deteet IgH-R. Namalwa cell line and U-937 cell line were used for positive and negative control respectively. The β-aetin gene was chosen as inter centred. DNA was isolated hy phenol-chlorform-isoamyl alcohol and then was amplified by SYBR Green RT-FQ-PCR targeting the IgH-R CDR m. Results： Mehing curve analysis could confirm the specificity of IgH-R. The positive rate deiceted by RT-FQ-PCR was 63.2%. The positive resuhs of IgH/β-actin were between 0. 01 and 4131.69. and the median was 0. 42. There was a significant difference between stage Ⅰ /Ⅱ and stage m/IV in IgH-R quantiiy （P = 0. 018）. Nonparametric test showed Ihal there was a significant difference between patients with normal LDH and patients with elevated LDH （ P =0. 046）. Conclusions： SYBR Green RT-FQ-PCR is a valuable, feasible and sensitive tool to detect IgH rearrangement in DI,BCI,. Detecting IgH-R using RT-FQ-PCR can help staging more accurately.

关 键 词：弥漫大B细胞淋巴瘤 免疫球蛋白重链基因 实时荧光定量PCR 嵌套式PCR 

分 类 号：R733.1[医药卫生—肿瘤]