摘要
目的探讨依布替尼克服弥漫大B细胞淋巴瘤（DLBCL）细胞耐药的机制。方法①体外实验：以DLBCL细胞系SUDHL10细胞（GCB亚型）、HBL-1（ABC亚型）以及8例DLBCL患者原代细胞为研究对象，与正常人骨髓基质细胞（MSC）共培养后，显微镜下计数向MSC趋化迁移及与MSC黏附的DLBCL细胞数，ELISA法检测MSC的CXCL12表达水平，流式细胞术检测DLBCL细胞的CXCR4表达水平；以携带有CXCR4的慢病毒转染HBL-1细胞，米托蒽醌、依布替尼处理后与MSC共培养，流式细胞术检测细胞凋亡水平；倒置显微镜下观察HBL．1细胞集落形成情况。②体内实验：以HBL．1细胞构建的NOD／SCID肿瘤模型小鼠为研究对象，观察依布替尼治疗后肿瘤体积变化。结果①依布替尼处理后，DLBCL细胞向MSC的迁移数和与MSC的黏附比例明显降低（P值均〈0．05），并呈剂量依赖性。②与依布替尼处理前比较，处理后MSC的CXCL12表达水平降低（SUDHL10细胞：660pg／ml对1400pg／ml，P=0．004；HBL-1细胞：720pg／ml对1490pg／ml，P=0．018；DLBCL原代细胞：850pg／ml对1450pg／ml，P=0．004），DLBCL细胞的CXCR4表达水平降低（P值均〈0，05）。③共培养时，对照组、米托蒽醌组、依布替尼组、米托蒽醌组＋依布替尼组的HBL．1细胞凋亡比例分别为15．1％、17．5％、23．5％、58．7％，转染过表达CXCR4后，HBL．1细胞凋亡比例分别为14．2％、16．1％、22．5％、38．3％，共培养联合用药组HBL．1细胞凋亡比例高于单药培养组，差异均有统计学意义（P值均〈0．05）。④对照组、MSC组、依布替尼组、MSC组＋依布替尼组集落数分别为113±5、205±4、62±9、123±3（每孔2．5×10^3），模型小鼠皮下肿瘤体积分别为6500、17000、4000、10000mm^3，依布替尼处理后较处理前集落数和肿瘤体积明显减少，差异均有统计学意义）P值均〈0．05）。结论依布替尼靶向作用于CXCL12�
Objective To explore the mechanism of ibrutinib on drug resistance diffuse large B-cell lymphoma （DLBCL） cells. Methods DLBCL cell line was cultured with mesenchymal stem cells （MSC）, and DLBCL cells which migrated and adhered to MSC under microscope was counted. The secretion of CXCL 12 by MSC were measured by ELISA. The expression of CXCR4 on DLBCL cells were measured by flow cytometry, HBL-1 cells were transfected with a CXCR4-lentivector. An Annexin V- binding assay was used to detect the induction of apoptosis. Clonogenic growth of DLBCL cells was evaluated on MethoCult media. Ibrutinib was injected into NOD/SCID mice, tumor growth was assessed via caliper measurements every 3 days. Results MSC promoted migration and adhesion of DLBCL cells to MSC. Ibrutinib inhibited migration and adhesion of DLBCL cells to MSC in a dose-dependent manner （P 〈 0.05）. CXCL 12 secreted by MSC and CXCR4 expressed on DLBCL cells could induce each other, which upgraded the levels of secretion and expression, lbrutinib could inhibit the secretion of CXCL12 （SUDHLI0：660 pg/ml vs 1 400 pg/ml, P = 0.004; HBL-1：720 pg/ml vs 1 490 pg/ml, P = 0.018; DLBCL： 850 pg/ml vs 1 450 pg/ml, P = 0.004） and expression of CXCR4 （P 〈 0.05）. When co-cultured with MSC, the ratio of HBL-1 cells apoptosis in the group of control, mitoxantrone, ibrutinib, mitoxantrone＋ibrutinib were respectively 15.1%, 17.5%, 23.5%, 58.7%. After transfected with a CXCR4-lentivector and overexpressed CXCR4, the ratios of HBL-1 cells apoptosis were 14.2%, 16.1%, 22.5%, 38.3% respectively. The ratio of DLBCL cells apoptosis induced by mitoxantrone was lower when co-cultured with MSC （P 〈 0.05）. But with the addition of ibrutinib, the ratio of apoptosis was increaed and it was similar to cultivation without MSC, which suggested ibrutinib could inhibit drug-resistance induced by MSC. But after transfected with a CXCR4-lentivector, the overexpression of CXCR4 was detected and the ratio of apoptosis was significantly lower when co-cultured
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2017年第12期1036-1042,共7页
Chinese Journal of Hematology
基金
国家自然科学基金（81600163、81570201）
