弥漫大B细胞淋巴瘤中存在巨噬细胞表型的肿瘤细胞并具有临床意义  

Presence and clinical significance of tumor cells with macrophage phenotype in diffuse large B-cell lymphoma

作　　者：李慧卉 张亮[1] 许周毅 王伟[1] 王哲[1,2] Li Huihui;Zhang Liang;Xu Zhouyi;Wang Wei;Wang Zhe(Department of Pathology,the First Affiliated Hospital of University of Science and Technology of China/Anhui Provincial Hospital,Hefei 230036,China;Department of Pathology,Xijing Hospital,Air Force Medical University,Xi’an 710032,China)

机构地区：[1]中国科学技术大学附属第一医院(安徽省立医院)病理科,合肥230036 [2]空军军医大学西京医院病理科,西安710032

出　　处：《临床与实验病理学杂志》2025年第2期162-170,共9页Chinese Journal of Clinical and Experimental Pathology

基　　金：安徽省高等学校科学研究项目(2023AH053402)。

摘　　要：目的探究弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)组织中具有巨噬细胞表型肿瘤细胞的存在、比例、临床意义和来源,探究能否在体外DLBCL细胞系中诱导CD68^(+)阳性肿瘤细胞。方法首先通过多重免疫荧光染色定性技术检测DLBCL样本中CD68^(+)CD163^(+)CD20^(+)PAX5^(-)细胞的存在,并检测CD79a^(+)B淋巴细胞、CD68^(+)巨噬细胞以及CD68^(+)CD79a^(+)双阳性细胞的比例。根据双阳性细胞的比例进行分组,探究CD68^(+)B淋巴细胞比例对DLBCL患者预后以及临床病理特征的影响。对具有BCL6基因断裂阳性的病例,使用免疫荧光与免疫原位杂交联合技术进行CD68与BCL6基因断裂共定位,判断具有巨噬细胞表型的B淋巴细胞的肿瘤性质。使用佛波酯(phorbol myristate acetate,PMA)处理DLBCL细胞系(OCI-LY3、SU-DHL2)诱导CD68^(+)肿瘤细胞,通过流式细胞术检测CD68、PAX5蛋白水平变化。采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术检测B细胞分化发育的重要转录因子PAX5及巨噬细胞相关基因(CD68、ARG1、CD163、CD206、Dectin-1、PU.1、C/EBPα、C/EBPβ)mRNA水平变化。此外,将PMA处理后的DLBCL细胞系(OCI-LY3、SU-DHL2)与pH敏感性荧光染料pHrodo共同孵育,以检测PMA处理后DLBCL细胞的吞噬能力。结果50例DLBCL中CD68^(+)B淋巴细胞比例为0~9.3%,总生存期0.0082~4.2年,CD68^(+)B淋巴细胞低表达组患者的总生存期显著低于CD68^(+)B淋巴细胞高表达组(P=0.039)。CD68^(+)B淋巴细胞比例不同分组间DLBCL的分子分型差异有统计学意义(P=0.0095)。DLBCL中CD68^(+)B淋巴细胞来源于肿瘤细胞。使用PMA处理DLBCL细胞后CD68^(+)细胞和CD68^(+)PAX5^(-)细胞比例显著上升(P<0.05)、其他巨噬细胞标志物CD68、ARG1、CD163、CD206、Dectin-1、PU.1、C/EBPα、C/EBPβ以及PAX5 mRNA相对表达量与对照组相比,差异有统计学意义(P<0.05)。PMA处理DLBCL细胞后细胞吞噬能力增强。结论DLBCL组织中存在一定比Purpose To investigate the presence,proportion,clinical significance and origin of tumor cells with a macrophage phenotype in tumor tissues of patients with diffuse large B-cell lymphoma(DLBCL),and to explore whether CD68 positive tumor cells can be induced in DLBCL cell lines in vitro.Methods The presence of CD68^(+)CD163^(+)CD20^(+)PAX5^(-)cells in the samples of DLBCL patients was first qualitatively detected by multiplex immunofluorescence staining,and then the proportion of CD79a^(+)B lymphocytes,CD68^(+)macrophages,and CD68^(+)CD79a^(+)double-positive cells were quantified.Patients were grouped according to the proportion of double-positive cells,and the differences in prognosis and clinicopathological features of DLBCL patients between subgroups were investigated.For cases with positive BCL6 gene locus breaks,co-localization of CD68 with BCL6 gene breakapart was performed using combined immunofluorescence and immunological in situ hybridization to ascertain the tumor nature of B cell with a macrophage phenotype.DLBCL cell lines(OCI-LY3,SU-DHL2)were treated with phorbol myristate acetate(PMA),and changes in the proteins levels of CD68 and PAX5 proteins were detected by flow cytometry.Quantitative real-time PCR(qRT-PCR)was used to detect mRNA levels of PAX5,an important transcription factor for B cell differentiation and development,and macrophage-related genes(CD68,ARG1,CD163,CD206,Dectin-1,PU.1,C/EBPα,C/EBPβ).In addition,PMA-treated DLBCL cell lines(OCI-LY3,SU-DHL2)were co-incubated with pH-sensitive fluorescent dye pHrodo to detect the phagocytosis ability of PMA-treated DLBCL cells.Results The percentage of CD68^(+)B lymphocytes in 50 patients with DLBCL varied from 0 to 9.3%,and the overall survival(OS)ranged from 0.0082 to 4.2 years.Patients with the low CD68^(+)B lymphocytes group exhibited a significantly lower OS compared to those in the high CD68^(+)B lymphocytes group(P=0.039).There was a significant difference in the molecular typing of DLBCL patients(P=0.0095)between different subgroups for th

关 键 词：弥漫大B细胞淋巴瘤 巨噬细胞表型B细胞 免疫荧光 

分 类 号：R733.4[医药卫生—肿瘤]