细胞衰老诱导的抑制性细胞因子累积对DLBCL细胞功能的影响

Effect of accumulation of inhibitory cytokines induced by cellular senescence on DLBCL cell function

目的探讨细胞衰老在弥漫大B细胞淋巴瘤(DLBCL)细胞抵抗化疗中的作用及机制。方法将入组的DLBCL患者分为初诊(ND)组、完全缓解(CR)组、复发/难治(r/r)组。分别应用HE染色和β-半乳糖苷酶染色法检测淋巴结反应性增生患者、r/r DLBCL患者淋巴结组织衰老情况;用β-半乳糖苷酶染色法检测ND、r/r组DLBCL患者外周血中的衰老情况;用10、20、40 nmol/L的阿霉素(DOX)反复诱导DLBCL细胞株LY8衰老,构建衰老模型;CCK-8法检测衰老模型细胞增殖情况;流式细胞术检测衰老模型细胞凋亡情况;ELISA法检测DLBCL患者血清和衰老模型上清液中细胞因子表达。结果r/r组DLBCL患者淋巴结组织及外周血中的衰老细胞明显增多且血清中白细胞介素(IL)-6、IL-8、IL-10、IL-35、转化生长因子-β1(TGF-β1)等促炎性和免疫抑制性细胞因子分泌增加;使用40 nmol/L DOX成功诱导LY8细胞衰老,衰老细胞比例24.77%。对对照组及DOX诱导组进行功能检测,结果提示衰老组细胞增殖率低于对照组但高于10 nmol/L和20 nmol/L DOX组(P<0.05);凋亡率高于对照组但低于10 nmol/L和20 nmol/L DOX组(P<0.05)。同时,各组IL-2、IL-6、IL-8、IL-10、TGF-β1分泌量差异有统计学意义(P<0.05),与对照组比较,衰老组细胞上清液中IL-2、IL-6、IL-8、IL-10、IL-35、TGF-β1等各种促炎和免疫抑制性细胞因子分泌增加。结论细胞衰老通过诱导抑制性细胞因子累积促进DLBCL细胞凋亡抵抗。

Objective To investigate the role and mechanism of cellular senescence in diffuse large B cell lymphoma(DLBCL)cells resistance to chemotherapy.Methods DLBCL patients in the group were divided into Newly Diagnosed(ND)group,Complete remission(CR)group,relapsed/refractory(r/r)group.HE staining and senescenceβ-galactosidase staining were used to detect the senescence of lymphatic tissue in the lymph node reactive hyperplasia patients and the r/r DLBCL patients;senescenceβ-galactosidase staining was used to detect the peripheral blood of DLBCL patients in the ND group and the r/r group;the expression of inflammatory factors in serum of DLBCL patients was detected by ELISA;an senescence model was constructed by repetitively induce senescence of DLBCL cell line LY8 with 10,20,and 40 nmol/L doxorubicin(DOX);CCK-8 was used to detect the cell proliferation of senescent model.Flow cytometry was used to detect the apoptosis of senescent model.ELISA was used to detect cytokine expression in serum of DLBCL patients and supernatant of senescence model.Results Senescent cells in lymph node tissues and peripheral blood of r/r DLBCL patients significantly increased,and the secretion of pro-inflammatory and immunosuppressive cytokines such as interleukin-6(IL-6),interleukin-8(IL-8),interleukin-10(IL-10),interleukin-35(IL-35)and transforming growth factorβ1(TGF-β1)in serum increased;40nmol DOX was used to induce senescence of LY8 cells,and the proportion of senescent cells was 24.77%.The function detect of control group and DOX induced group showed that the cell proliferation rate of the senescence group was lower than that of the control group but higher than that of the 10 nmol/L and 20 nmol/L DOX groups(P<0.05);The apoptosis rate was higher than the control group but lower than the 10 nmol/L and 20 nmol/L DOX groups(P<0.05).Meanwhile,IL-2,IL-6,IL-8,IL-10,TGF-β1 secretion levels in each group were significantly different(P<0.05).Compared with the control group,the secretion of various pro-inflammatory and immunosuppressive cytokines such as IL-2,IL-6,IL-8,IL-10,IL-35,TGF-β1 in the supernatant of the senescent group increased.Conclusion Cellular senescence promotes DLBCL cell apoptosis resistance by inducing inhibitory cytokine accumulation.