应用CpG-寡脱氧核苷酸免疫刺激进行慢性淋巴细胞白血病的染色体研究  

Chromosome study on chronic lymphocytic leukemia using CpG-oligodeoxynucleotide as immunostimulant agent

作　　者：吴亚芳[1] 薛永权[1] 陈苏宁[1] 姚利[1] 蒋慧[1] 张俊[1] 沈娟[1] 潘金兰[1] 王勇[1] 白淑潇[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所,215006

出　　处：《中华医学遗传学杂志》2010年第1期86-91,共6页Chinese Journal of Medical Genetics

摘　　要：目的验证CpG-寡脱氧核苷酸（CpG—oligodeoxynucleotide，CpG—ODN）免疫刺激是否能提高B细胞慢性淋巴细胞白血病（chronic lymphocytic leukemia，CLL）核型异常检出率。方法抽取57例B-CLL患者的骨髓或外周血，分别加入CpG-ODN DSP30＋白细胞介素-2（interleukin-2，IL-2）、植物血凝素（phytohemagglutinin，PHA）、美洲商陆（pokeweed，PWM）和IL-2后进行培养，5d后按常规收获并制备染色体，应用R显带技术进行核型分析。应用Cen12、D13S25、Rb1、ATM、P53、MYB和IgH探针对其中19例核型正常的患者进行组合荧光原位杂交（fluorescence in situ hybridization，FISH）检测。抽提骨髓或外周血单个核细胞基因组DNA，应用聚合酶链反应（polymerase chain reaction，PCR）扩增免疫球蛋白重链可变区（immunoglobulin variable heavy chain，IgVH）并进行DNA序列分析。应用流式细胞仪（flow cytometry，FCM）检测白血病细胞的CD38和ZAP70表达。结果DSP30＋IL-2组的异常核型检出率（43．85％）明显高于PHA组（15．09％）、PWM组（17．31％）和IL-2组（3．13％）（P〈0．01）。DSP30＋IL-2组共检出畸变52种。数目异常以＋12或＋12伴其它异常为最多见（9例）；结构异常以易位为最多见（14例），包括平衡易位11例和不平衡易位3例，前者中又以14q32重排为最多见（7例）。FISH显示19例正常核型患者中13例（68．42％）显示1种或多种异常，包括12三体和p53缺失各1例，IgH重排和部分缺失各1例，13q14．3缺失11例，其中5例合并RB1缺失，1例合并RB1部分缺失，未见ATM和MYB缺失者。PCR揭示10／18例有IgVH突变（55．55％）。FCM检测揭示10／45例CD38＋，35／45例CD38-，11／27例ZAP70＋，16／27例ZAP70-。同时进行CD38和ZAP70检测的26例中，5例为CD38＋ZAP70＋，13例为CD38-ZAP70-，2例为CD38＋ZAP70-，6例为CD38-ZAP70＋。统计学分析揭示复杂核型异常与IgVH无突变之间有相关性，但�Objective To investigate whether CpG-oligodeoxynucleotide （CpG-ODN） can improve the detection rate of the karyotypic abnormalities in chronic lymphocytic leukemia （CLL）. Methods The bone marrow （BM） or peripheral blood （PB） cells from 57 cases of CLL were collected and cultured with CpG-ODN DSP30 ＋ interleukin-2 （IL-2）, phytohemagglutinin （ PHA）, pokeweed （PWM） or IL-2, respectively. Five days later cells were harvested for chromosome preparation. Karyotypic analysis was done using R banding technique. Panel fluorescence in situ hybridization （FISH） was carried out on 19 cases of CLL with normal karyotypes using the following probes： Cen12, D13S25, Rbl, ATM, p53, MYB and IgH. Genomic DNA from 21 cases of them was extracted from BM or PB leukocytes. The immunoglobulin variable heavy chain （IgVH） was amplified by polymerase chain reaction （PCR） and sequenced. CD38 and ZAP70 expressions in the leukemic cells were determined by flow eytometry （FCM）. Results The detection rate of karyotypic abnormalities in the CpG-ODN ＋IL-2 group （43. 85 %） was obviously higher than that in thePHA （15.09%）, PWM （17.31%） and IL-2 （3.13%） groups （P〈0.01）. Fihy-twotypes of karyotypie abnormalities were found. Among them, trisomyl2 （＋ 12） or ＋ 12 with other abnormalities were the most common, while translocations were the most frequent structural abnormalities including 3 unbalanced and 11 balanced translocations, among them 7 had rearrangements involving 14q32. Thirteen cases showed one or more abnormalities on FISH including trisomy 12 and p53 deletion each in one case, IgH rearrangement and partial deletion each in one ease, 13q4.3 deletion in 11 cases of which 5 cases also had Rbl deletion, 1 case had Rbl partial deletion. No case with ATM or MYB deletions was found. PCR detected IgVH mutations in 10/21 cases. FCM showed 10/45 cases were CD38 positive, but 35 /45 were CD38 negative, 11/27 cases expressed ZAP70, but 16/27 did not. Among the 26 cases examin

关 键 词：慢性淋巴细胞白血病 细胞遗传学 CpG-寡脱氧核苷酸 荧光原位杂交 免疫球蛋白重链可变区 突变 

分 类 号：R733.7[医药卫生—肿瘤]