CpG-寡核苷酸免疫刺激法在慢性淋巴细胞白血病遗传学研究中的应用

目的:探索CpG-寡核苷酸(CpG-oligodeoxynucleotide,CpG-ODN)免疫刺激法在慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)细胞染色体培养中的价值,对比国内外相关研究的差异,进一步提高CLL核型培养成功率及异常核型检出率。方法:收集82例CLL患者的骨髓标本,分别应用植物血凝素(PHA)、CpG-寡核苷酸联合白介素-2(CpG-ODN DSP30+IL-2)刺激培养72 h后收获细胞,制备染色体,进行常规细胞遗传学(conventional cytogenetics,CC)分析。同时应用D13S25、Rb1、ATM、p53和CSP12探针进行间期荧光原位杂交(interphase fluorescence in situ hybridization,iFISH)检测。比较2种细胞刺激剂进行染色体培养及iFISH检测结果的差异性。结果:PHA与CpG-ODN DSP30+IL-22种免疫刺激法核型培养成功率(可分析分裂相t≥20个)分别为90.2%(74例)和68.3%(56例),异常核型检出率分别为13.5%(10例)和46.4%(26例)。PHA组核型培养成功率显著高于CpG-ODN DSP30+IL-2组(P=0.01),而CpG-ODN DSP30+IL-2组异常核型检出率显著高于PHA组(P=0.003)。iFISH异常核型检出率(74.4%,61例)显著高于CpG-ODN DSP30+IL-2组(P=0.000)。iFISH检测能验证CC分析检出的异常。结论:应用CpG-ODN DSP30+IL-2免疫刺激法培养CLL细胞,可提高异常核型检出率,特别是能检出提示预后差的各种易位。

Application of CpG Oligodeoxynucleotide Immunostimulation in Chromosome Study of Chronic Lymphoblastic Leukemia

Objective:To explore the value of CpG-oligonucleotide(CpG-ODN)immunostimulatory method in chromosome culture of chronic lymphocytic leukemia(CLL)cells and to compare the differences between related studies at home and abroad,so as to improve the success rate of CLL karyotype culture and the detection rate of abnormal karyotypes.Methods:Bone marrow samples from 82 CLL patients were collected and cultured with phytohemagglutinin(PHA),CpG-oligonucleotide plus interleukin-2(CpG-ODN DSP30+IL-2)for 72 hours.Chromosomes were prepared and analyzed by conventional cytogenetics(CC).Meanwhile,D13 S25,Rb1,ATM,p53 and CSP12 probes were used for interphase fluorescence in situ hybridization(iFISH)test.The differences of chromosome culture and iFISH test results between two cell stimulants were compared.Results:The success rate of karyotype culture in PHA and CpG-ODN DSP30+IL-2 immunostimuli(analyzable mitotic t>20)was 90.2%(74 cases),68.3%(56 cases)respectively,and the detection rate of abnormal karyotype was 13.5%(10 cases)and 46.4%(26 cases),respectively.The success rate of karyotype culture in PHA group was significantly higher than that in CpG-ODN DSP30+IL-2 group(P=0.01).The detection rate of abnormal karyotypes in CpG-ODN DSP30+IL-2 group was significantly higher than that in PHA group,and the difference was statistically significant(P=0.003).The detection rate of abnormal karyotypes in iFISH group was 74.4%(61 cases),which was significantly higher than that in CpG-ODN DSP30+IL-2 group(P=0.000).iFISH detection could verify the abnormalities detected by CC analysis.Conclusion:Application of CpG-ODN DSP30+IL-2 immunostimulation method in culture of CLL cells can enhance the detection rate of abnormal karyotypes,especially the detection of various translocations suggesting poor prognosis.