依维莫司联合吉西他滨对弥漫大B细胞淋巴瘤的协同抗肿瘤作用  

Synergistic Antitumor Effect of Everolimus Combined with Gemcitabine on Diffuse Large B-Cell Lymphoma

作　　者：左休琴 谭春莲 李晓明 马涛 ZUO Xiu-Qin;TAN Chun-Lian;LI Xiao-Ming;MA Tao(Department of Hematology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属医院血液内科,四川泸州646000

出　　处：《中国实验血液学杂志》2023年第1期81-88,共8页Journal of Experimental Hematology

基　　金：西南医科大学校级基金项目(2017-ZRQN-091)。

摘　　要：目的:探讨mTOR抑制剂依维莫司与吉西他滨对弥漫大B细胞淋巴瘤U2932细胞株的增殖、凋亡以及细胞周期的影响,并进一步研究其分子机制,为DLBCL的临床治疗提供新的思路和实验依据。方法:采用CCK-8法检测依维莫司、吉西他滨对U2932细胞增殖的影响,计算两药的IC50,利用Compu Syn软件计算两药的联合指数(CI)。Annexin V-FITC/PI双染色法流式细胞术检测依维莫司和吉西他滨对U2932细胞凋亡的影响。碘化丙啶(PI)单染色流式细胞分析法检测依维莫司和吉西他滨对U2932细胞周期的影响。Western blot法检测依维莫司和吉西他滨对通道蛋白p-m TOR、p-4EBP1,抗凋亡蛋白MCL-1、Survivin,细胞周期蛋白Cyclin D1的影响。结果:依维莫司和吉西他滨对U2932细胞的增殖抑制作用具有时间-剂量效应(r=0.465,0.848;0.555,0.796)。根据Compu Syn软件计算,依维莫司联合吉西他滨抑制U2932细胞增殖在24 h、48 h和72 h的CI<1,具有协同作用。10 nmol/L依维莫司、250 nmol/L吉西他滨单药及联合处理U2932细胞48 h后,依维莫司和吉西他滨均可诱导细胞凋亡,与对照组比较差异有统计学意义(P<0.05)。依维莫司联合吉西他滨作用细胞后凋亡率较单药明显增强(P<0.05)。依维莫司、吉西他滨单药及联合用药与对照组比较均使处于G1期的细胞比例明显升高(P<0.05)。联合用药时,处于G1期的细胞比例明显更高(P<0.05)。依维莫司联合吉西他滨组可以显著下调p-m TOR及效应蛋白p-4EBP1的表达,同时下调抗凋亡蛋白MCL-1、Survivin及周期蛋白Cyclin D1的表达(P<0.05)。结论:依维莫司和吉西他滨可协同抑制U2932细胞的增殖,其机制可能通过下调MCL-1、Survivin蛋白表达协同诱导细胞凋亡,通过下调Cyclin D1蛋白表达协同阻滞细胞周期进展。Objective:To investigate the effects of m TOR inhibitors everolimus (EVE) and gemcitabine (GEM) on the proliferation,apoptosis and cell cycle of diffuse large B-cell lymphoma (DLBCL) cell line U2932,and further explore the molecular mechanisms,so as to provide new ideas and experimental basis for the clinical treatment of DLBCL.Methods:The effect of EVE and GEM on the proliferation of U2932 cells was detected by CCK-8 assay,the IC50 of the two drugs was calculated,and the combination index (CI) of the two drugs was calculated by Compu Syn software.The effect of EVE and GEM on apoptosis of U2932 cells was detected by flow cytometry with Annexin V-FITC/PI staining.Flow cytometry with propidium iodide (PI) staining was used to detect the effect of EVE and GEM on the cell cycle of U2932cells.Western blot assay was used to detect the effects of EVE and GEM on the channel proteins p-m TOR and p-4EBP1,the anti-apoptotic proteins MCL-1 and Survivin,and the cell cycle protein Cyclin D1.Results:Both EVE and GEM could significantly inhitbit the proliferation of U2932 cells in a time-and dose-dependent manner (r=0.465,0.848;0.555,0.796).According to the calculation of Compu Syn software,EVE combined with GEM inhibited the proliferation of U2932 cells at 24,48 and 72 h with CI<1,which had a synergistic effect.After treated U2932 cells with 10 nmol/L EVE,250 nmol/L GEM alone and in combination for 48 h,both EVE and GEM induced apoptosis,and the difference was statistically significant compared with the control group (P<0.05).The apoptosis rate was significantly enhanced after EVE in combination with GEM compared with single-agent (P<0.05).Both EVE and GEM alone and in combination significantly increased the proportion of cells in G1phase compared with the control group (P<0.05).The proportion of cells in G1phase was significantly increased when the two drugs were combined (P<0.05).The expression of p-m TOR and effector protein p-4EBP1 was significantly downregulated in the EVE combined with GEM group,the expression of anti-apo

关 键 词：MTOR抑制剂 依维莫司 吉西他滨 弥漫大B细胞淋巴瘤 

分 类 号：R733.4[医药卫生—肿瘤]