利妥昔单抗与依鲁替尼对弥漫性大B细胞淋巴瘤细胞增殖和凋亡协同作用研究

目的 近年来发现依鲁替尼是B细胞恶性肿瘤的新型靶向药物,利妥昔单抗联合新药依鲁替尼对弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)的研究逐步展开,本研究探讨利妥昔单抗与依鲁替尼对DLBCL细胞系Farage增殖及凋亡影响的协同作用.方法 不同浓度的单药利妥昔单抗和单药依鲁替尼处理Farage细胞24、48和72 h后,采用CCK8法检测细胞增殖抑制率.15 μmol/L依鲁替尼处理Farage细胞48 h后,采用RT-PCR法检测凋亡相关因子mRNA的表达变化.含人补体的培养条件下,1 μg/mL利妥昔单抗联合10 μmol/L依鲁替尼同时处理Farage细胞48 h后,采用CCK8法检测增殖抑制率,采用流式细胞术检测凋亡率.结果 利妥昔单抗在不含人补体的培养基中对Farage细胞增殖抑制作用不明显,在含人补体的培养基中有明显增殖抑制作用,并呈浓度和时间依赖性.依鲁替尼在含或不含人补体的培养条件下对Farage细胞均有明显增殖抑制作用,呈浓度和时间依赖性,2种条件下的增殖抑制率差异无统计学意义(F=0.978,P=0.329),15 μmol/L依鲁替尼作用Farage细胞48 h时,Fas(1.63±0.09)、Caspase-8(1.90±0.11)和Caspase-3(2.20±0.11)mRNA相对表达量均高于空白组(1.00±0.00).在含人补体的培养基中,1 μg/mL利妥昔单抗联合10 μmol/L依鲁替尼同时处理Farage细胞48 h后,联合组增殖抑制率为(57.06±1.48)%,高于依鲁替尼单药组(33.83±3.39)%和利妥昔单抗单药组(26.92±2.74)%,差异有统计学意义,F=87.403,P<0.001.联合组的凋亡和坏死率(44.30±2.20)%高于利妥昔单抗单药组(21.73±2.00)%和依鲁替尼单药组(17.44±1.60)%,且利妥昔单抗单药组和依鲁替尼单药组均高于空白组(2.32±0.21)%, 差异均有统计学意义,F=327.205,P<0.001.结论 利妥昔单抗可通过CDC效应引起DLBCL细胞凋亡坏死及增殖抑制,依鲁替尼可能通过上调Fas、Caspase-3和Caspase-8基因的表达,促使DLBCL细胞凋亡和增殖抑制,利妥昔单抗联合依鲁替尼对DLBCL细胞的增殖抑制和凋亡坏死作用明显强于单药利妥昔单抗或依鲁替尼.

Effect of Rituximab combination with ibrutinib on proliferation and apoptosis of diffuse large B cell lymphoma cell lines

OBJECTIVE A part of patients of diffuse large B cell lymphoma after Rituximab combined chemotherapy as first-line therapy still have relapse.Ibrutinib is a new type of targeted drugs in B cell malignancies.The aim of this study was to investigate the effect of rituximab combination with ibrutinib on cell proliferation and apoptosis of diffuse large B cell lymphoma cell lines Farage.METHODS The Farage cells were treated with various concentrations of rituximab for 24,48,72 h, the inhibitory rate of cell proliferation was measured by CCK8 assay.The Farage cells were treated with 15 μmol/L of ibrutinib for 48 h,the mRNAs of apoptosis factors were measured by RT-PCR.The Farage cells were treated with 1 μg/mL rituximab and 10 μmol/L ibrutinib concurrently in medium containing human complement for 48 h, the inhibitory rate of cell proliferation was measured by CCK8 assay, the apoptosis and death rate of cell were detected by flow cytometry.RESULTS The growth of farage cells had not obvious inhibition in medium excluding human complement,whereas it was inhibited obviously in concentration-dependence and time-dependence manners after treated with rituximab.The growth of farage cells was inhibited in concentration-dependence and time-dependence manners after treated with ibrutinib,and the inhibition of cells was no significantly difference between that in medium with or without human complement (F=0.978,P=0.329).The Farage cells were treated with 15 μmol/L of ibrutinib for 48 h,and the mRNA expression levels of Fas (1.63±0.09)(t=7.181,P=0.019),Caspase-8 (1.90±0.11)(t=7.824,P=0.016),Caspase-3 (2.20±0.11)(t=10.392,P=0.009)were higher than that of control group (1.00±0.00).The Farage cells were treated with 1 μg/mL rituximab and 10 μmol/L ibrutinib concurrently in medium containing human complement for 48 h, and the inhibition rate (57.06±1.48)% of combination group were higher than that of ibrutinib group (33.83±1.96)% and rituximab group (26.96±1.58)%,F=87.403,P<0.001.Apoptosis and death rate of combination group (44.30±2.20)% were higher than that of ibrutinib group (17.44±1.60)% and rituximab group (21.73±2.20)%,that in ibrutinib group and rituximab group also higher than in control group (2.32±0.21)%, F=327.205,P<0.001.CONCLUSIONS Rituximab can inhibit the proliferation,apoptosis and necrosis of diffuse large B cell lymphoma cell lines mainly through complement dependent cytotoxicity, ibrutinib can inhibit the proliferation and apoptosis of diffuse large B cell lymphoma cell lines mainly through up-regulated apoptosisgene such as Fas, Caspase-8,Caspase-3.The combination of rituximab with ibrutinib has stronger effect of inhibition, apoptosis and necrosis on diffuse large B cell lymphoma cell lines compared with the single use of rituximab or ibrutinib.