BET抑制剂对弥漫性大B细胞淋巴瘤细胞生长及外周血Th17细胞数量和相关细胞因子表达的影响

目的:探讨溴结构域和超末端结构域（bromodomain and extra terminal domain，BET）抑制剂对弥漫性大B细胞淋巴瘤CRL-2630细胞生长的影响，以及对弥漫性大B细胞淋巴瘤BALB/c-nu裸鼠外周血中辅助性T细胞17（helper T cells,Th17）数量和相关细胞因子表达的影响。方法:培养弥漫性大B细胞淋巴瘤株CRL-2630，使用不同浓度BET抑制剂（2、4、8、16、32 nmol/L）处理48 h，32 nmol/L BET抑制剂处理不同时间（12、24、36、48 h），CCK-8法检测各处理细胞活性；集落形成实验检测不同BET抑制剂浓度处理后细胞集落形成能力；Annexin V-FITC/PI双染法检测不同BET抑制剂浓度处理后细胞凋亡情况；实时荧光定量PCR与Western blot检测32 nmol/L BET抑制剂处理CRL-2630细胞48 h后HMGA1 mRNA与蛋白的表达水平；构建HMGA1过表达载体并通过脂质体介导法转染CRL-2630细胞，并用32 nmol/L BET抑制剂处理48 h，检测细胞活性与凋亡情况；构建裸鼠弥漫性大B细胞淋巴瘤模型并采集外周血，流式细胞术检测Th17细胞比例，ELISA法检测相关细胞因子的含量。结果:在一定范围内，BET抑制剂呈剂量依赖性地抑制CRL-2630细胞的活性，32 nmol/L BET抑制剂以时间依赖性地抑制CRL-2630细胞的活性。随着BET抑制剂处理浓度的增高，CRL-2630细胞集落形成能力逐渐下降，凋亡率逐渐升高。32 nmol/L BET抑制剂处理CRL-2630细胞48 h后，细胞中HMGA1的mRNA和蛋白水平均明显下降。pcDNA3.4-HMGA1转染CRL-2630细胞再使用BET抑制剂处理后，细胞的活性升高而凋亡率明显下降。弥漫性大B细胞淋巴瘤裸鼠经BET抑制剂作用后，外周血Th17细胞比例和IL-6、IL-17、IL-23含量较生理盐水组明显下降。结论:BET抑制剂能有效抑制 CRL-2630细胞活性，并诱导其凋亡，在一定范围内呈时效和量效关系。BET抑制剂还可以抑制弥漫性大B细胞淋巴瘤裸鼠外周血Th17细胞数量和相关细胞炎性因子的分泌，其作用机制可能与HMGA1的表达下调有关。

Effects of BET inhibitor on the growth of diffuse large B-cell lymphoma cells and the expression of Th17 cells and related cytokines in peripheral blood

Objective:To investigate the effects of BET inhibitor on the growth of diffuse large B-cell lymphoma CRL-2630 cells and the expression of helper T cells 17(Th17) and related cytokines in peripheral blood of diffuse large B-cell lymphoma BALB/c-nu nude mice.Methods:Diffuse large B-cell lymphoma strain CRL-2630 was cultured,and CRL-2630 cells were treated with different concentrations of BET inhibitor(2,4,8,16,32 nmol/L) for 48 h,32 nmol/L BET inhibitor treatment of CRL-2630 cells at different times(12,24,36,48 h).CCK-8 assay was used to detect the activity of each treated cell.Colony formation assay was used to detect cell colony formation ability after treatment with different BET inhibitor concentrations.Annexin V-FITC/PI double staining method was used to detect apoptosis after treatment with different BET inhibitor concentrations.The expression of HMGA1 mRNA and protein was detected by Real-time PCR and Western blot detection of CRL-2630 cells treated with 32 nmol/L BET inhibitor for 48 h.The HMGA1 overexpression vector was constructed and transfected into CRL-2630 cells by liposome-mediated method,and treated with 32 nmol/L BET inhibitor for 48 h to detect cell activity and apoptosis.A diffuse large B-cell lymphoma model of nude mice was constructed and peripheral blood was collected.The proportion of Th17 cells was detected by flow cytometry,and the content of related cytokines was detected by ELISA.Results:Within a certain range,the BET inhibitor inhibited the activity of CRL-2630 cells in a dose-dependent manner,and the 32 nmol/L BET inhibitor inhibited the activity of CRL-2630 cells in a time-dependent manner.With the increase of the concentration of BET inhibitor treatment,the colony forming ability of CRL-2630 cells gradually decreased,and the apoptotic rate gradually increased.After treated with 32 nmol/L BET inhibitor for 48 h,the mRNA and protein levels of HMGA1 in the cells decreased significantly.When pcDNA3.4-HMGA1 was transfected into CRL-2630 cells and treated with BET inhibitor,the activity of the cells increased and the apoptotic rate decreased significantly.The proportion of Th17 cells and IL-6,IL-17 and IL-23 in peripheral blood decreased significantly in nude mice with diffuse large B-cell lymphoma.Conclusion:BET inhibitor can effectively inhibit the activity of CRL-2630 cells and induce apoptosis,which is aging and dose-effect in a certain range.BET inhibitor can also inhibit the number and correlation of Th17 cells in diffuse large B-cell lymphoma nude mice.The secretion of cellular inflammatory factors may be related to the down-regulation of HMGA1 expression.