阿卡替尼增强利妥昔单抗生物类似药HLX01对非GCB型DLBCL耐药细胞株的杀伤效果  

Acalabrutinib enhances killing effect of rituximab biosimilar HLX01 on rituximab-resistant non-GCB DLBCL cell line

作　　者：申晗 高冠论 许艳丽 张星 杜庆华 李庆山 SHEN Han;GAO Guan-lun;XU Yan-li;ZHANG Xing;DU Qing-hua;LI Qing-shan(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Hematology,Guangzhou Red Cross Hospital,Jinan University,Guangzhou 510220,China;Department of Hematology,the Second Affiliated Hospital of South China University of Technology,Guangzhou 510180,China)

机构地区：[1]华南理工大学医学院,广东广州510006 [2]暨南大学附属广州红十字会医院血液科,广东广州510220 [3]华南理工大学附属第二医院血液科,广东广州510180

出　　处：《中国病理生理杂志》2022年第1期11-19,共9页Chinese Journal of Pathophysiology

基　　金：广州市科技计划项目(No.201707010279)。

摘　　要：目的:探索阿卡替尼(Aca)联合利妥昔单抗(RTX)生物类似药HLX01增强对耐RTX的非生发中心B细胞样(非GCB型)弥漫大B细胞淋巴瘤(DLBCL)细胞株杀伤效果的相关机制。方法:(1)采用RTX浓度梯度递增结合大剂量间断冲击的方法在体外构建耐RTX的人非GCB型DLBCL细胞株NU-DUL-1-R。流式细胞术检测RTX补体依赖的细胞毒性(CDC)及抗体依赖的细胞介导的细胞毒性(ADCC)作用下耐药株和亲本株的死亡率,并检测亲本株和耐药株的生长曲线、细胞周期及表面抗原CD20、CD55和CD59表达。(2)用浓度为2.5、5、10、20、40和80μmol/L的单药Aca分别处理NU-DUL-1-R细胞48 h后,采用CCK-8法检测细胞活力抑制率,并计算半数抑制浓度(IC50)。再以20%新鲜正常人血清为补体来源,羧基荧光素琥珀酰亚胺酯(CFSE)标记肿瘤细胞后建立外周血单个核细胞与肿瘤细胞(效靶比为5∶1)体系,采用7-氨基放线菌素D(7-AAD)流式染色检测Aca联合RTX处理NU-DUL-1-R细胞后CDC及ADCC效应诱导的细胞死亡率,ELISA法检测白细胞介素17(IL-17)的表达,流式细胞术分析Th17细胞的比例。结果:(1)RTX对耐药株的CDC及ADCC效应均减弱;与亲本株相比,耐药株生长速度减慢,细胞周期分布改变,G1期细胞数目增加,S期细胞数目减少(P<0.05),CD20表达下调(P<0.05),但CD55及CD59表达无显著差异(P>0.05)。(2)Aca对NU-DUL-1-R细胞的IC50为12.51μmol/L;Aca与RTX联用组对NU-DUL-1-R细胞的CDC及ADCC作用均强于单药组(P<0.05)。Aca处理后Th17细胞比例降低,IL-17的分泌减少;与单用RTX组相比,Aca与RTX联用后Th17细胞比例和IL-17分泌水平均显著降低(P<0.05)。结论:耐RTX的人非GCB型DLBCL细胞株NU-DUL-1-R较亲本株生长速度减慢,细胞周期分布改变,表面CD20抗原表达减少;Aca增强了RTX生物类似药HLX01对非GCB型DLBCL耐药细胞株的杀伤作用,其机制可能与Aca减少IL-17的分泌有关。AIM:To explore the mechanism that combination of acalabrutinib(Aca)and rituximab(RTX) biosimilar HLX01 enhances the killing effect on RTX-resistant human non-germinal center B-cell-like diffuse large B-cell lymphoma(non-GCB DLBCL)cell line.METHODS:(1)Stepwise-increasing dose of RTX combined with high-dose intermittent pulse was used to establish the RTX-resistant human non-GCB DLBCL cell line NU-DUL-1-R. Flow cytometry was used to detect the complement-dependent cytotoxicity(CDC) and antibody-dependent cell-mediated cytotoxicity(ADCC)of RTX on RTX-resistant cells and parental cells. The growth curve,cell cycle and expression of CD20,CD55 and CD59 of the two cell lines were determined.(2)The NU-DL-1-R cells were treated with Aca for 48 h at the concentrations of 0,2. 5,5,10,20,40 and 80 μmol/L,the inhibitory rates of cell viability were detected by CCK-8 method,and the half inhibitory concentration(IC50)was calculated. After 20% fresh normal human serum was used as the source of complement,carboxyfluorescein succinimidyl ester(CFSE)-labeled tumor cells were co-cultured with peripheral blood mononuclear cells at a ratio of 1∶5,and then 7-aminoactinomycin D(7-AAD)flow cytometric staining was used to detect the CDC and ADCC of Aca combined with RTX on the tumor cells. ELISA was used to detect the secretion of interleukin-17(IL-17),and flow cytometry was used to analyze the percentage of Th17 cells.RESULTS:(1)Compared with the parental cell line,the CDC and ADCC of RTX on RTX-resistant cell line were significantly reduced(P<0. 05),and the RTX-resistant cells had a slower growth rate. The cell cycle distribution was changed,as indicated by the increased number of the cells in G1 phase and the decreased one in S phase. The CD20 expression was lowered in the RTX-resistant cell line(P<0. 05),while the expression of CD55 and CD59 was not significantly different between the two cell lines(P>0. 05).(2)The IC50 of Aca for NU-DUL-1-R cells was 12. 51 μmol/L. The CDC and ADCC effects on the NU-DUL-1-R cells in combination(R

关 键 词：利妥昔单抗 生物类似药 非生发中心B细胞样弥漫大B细胞淋巴瘤 阿卡替尼 TH17细胞 白细胞介素17 

分 类 号：R733.4[医药卫生—肿瘤] R363.2[医药卫生—临床医学]