【作者】 郭晶晶；

【导师】 夏凌辉；

【作者基本信息】 华中科技大学 ， 内科学（血液内科）， 2023， 硕士

【摘要】 目的:R-CHOP(利妥昔单抗联合环磷酰胺、阿霉素、长春新碱和泼尼松)是弥漫性大B细胞淋巴瘤(DLBCL)的一线治疗方案。大约30%~40%的DLBCL患者在治疗后难以治愈或复发。因此,寻找新的有效的治疗药物是提高DLBCL患者生存的迫切需要。槐耳,一种传统中药,已在其他几种类型的癌细胞中显示出较好的疗效,但在淋巴瘤中的作用尚未报道。本研究拟探讨槐耳对弥漫性大B细胞淋巴瘤的影响。方法:以生发中心B细胞/GCB(SU-DHL-4、SU-DHL-6)和活化B细胞/ABC(SUDHL-2)两种弥漫性大B细胞淋巴瘤亚型为研究对象。采用网络药理学分析槐耳治疗弥漫性大B细胞淋巴瘤的可行性以及可能的作用靶点和信号通路;CCK8法用于检测细胞活性;PI/Rnase染色法用于检测细胞周期;细胞凋亡采用Annexin V-FITC/PI双染法检测;线粒体膜电位的变化情况用JC-1荧光探针检测;特定蛋白的表达情况用蛋白质印迹分析检测。建立NOD-SCID小鼠SU-DHL-6和SU-DHL-2细胞异种移植瘤模型,观察槐耳在体内对弥漫性大B细胞淋巴瘤的作用。结果:槐耳治疗弥漫性大B细胞淋巴瘤的网络药理学分析结果显示AKT1,SRC,EGFR,JUN,CASP3可能在槐耳治疗DLBCL中具有重要作用,并且槐耳可能调控弥漫性大B细胞淋巴瘤发生发展中的多条重要的信号通路,包括PI3K-Akt信号通路,MAPK信号通路,NF-kappa B信号通路,P53信号通路,TNF信号通路,BCR信号通路等。体外实验证实槐耳可呈剂量依赖性地抑制GCB-DLBCL细胞系(SUDHL-4、SU-DHL-6)和ABC-DLBCL细胞系(SU-DHL-2)的细胞活力,引起GCBDLBCL细胞和ABC-DLBCL细胞的G1期细胞周期阻滞,并可通过激活caspase和抑制抗凋亡蛋白的表达诱导GCB-DLBCL细胞和ABC-DLBCL细胞的凋亡。此外槐耳还可显著下调参与DLBCL进展的分子并可增强BTK抑制剂(伊布替尼,奥布替尼,泽布替尼)对GCB-DLBCL细胞和ABC-DLBCL细胞的抑制作用。在体内实验中,在人GCB-DLBCL和ABC-DLBCL异种移植模型中槐耳可显著抑制肿瘤的生长。结论:上述结果显示,槐耳抑制GCB-DLBCL细胞和ABC-DLBCL细胞的增殖并诱导细胞凋亡,同时可增强BTK抑制剂(伊布替尼,奥布替尼,泽布替尼)对GCBDLBCL细胞和ABC-DLBCL细胞的抑制作用,提示槐耳可能为DLBCL药物治疗提供新的策略。更多还原

【Abstract】 Purpose: R-CHOP is the first-line therapy for diffuse large B-cell lymphoma(DLBCL).Approximately 30%~40% of patients with DLBCL are either refractory to cure or relapse after treatment.Therefore,finding new effective therapeutic agents is urgently needed to improve the survival of DLBCL patients.Huaier,a traditional Chinese medicine,has shown good efficacy in several other types of cancer cells.This study aims to investigate the effect of Huaier on DLBCL.Methods: Two diffuse large B-cell lymphoma subtypes,germinal center B cell/GCB(SU-DHL-4,SU-DHL-6)and activated B cell /ABC(SU-DHL-2),were used in this study.Network pharmacology was used to analyze the feasibility,potential targets and signaling pathways of Huaier in the treatment of DLBCL.CCK8 assay was used to detect cell viability.PI/Rnase staining was used to detect cell cycle.To detect cell apoptosis,Annexin V-FITC/PI double staining was used.JC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential.And to detect the expression of specific proteins,Western blot analysis was used.NOD-SCID xenograft models of SU-DHL-6 and SU-DHL-2 cells were established to observe the effect of Huaier in DLBCL in vivo.Results: Network pharmacology analysis showed that AKT1,SRC,EGFR,JUN,and CASP3 may play an important role in the treatment of DLBCL by Huaier,and Huaier may regulate multiple important signaling pathways in the occurrence and development of DLBCL,including PI3K-Akt signaling pathway.MAPK signaling pathway,NF-kappa B signaling pathway,P53 signaling pathway,TNF signaling pathway,BCR signaling pathway,etc.In vitro experiments,Huaier could effectively inhibit the cell viability of GCB-DLBCL cell lines(SU-DHL-4,SU-DHL-6)and ABC-DLBCL cell lines(SU-DHL-2)in a dosedependent manner,cause cell cycle arrest at G1 phase of GCB-DLBCL and ABC-DLBCL cells,and induce apoptosis of GCB-DLBCL and ABC-DLBCL cells by activating caspase and inhibiting the expression of anti-apoptotic proteins.In addition,Huaier could significantly down-regulate the molecules involved in DLBCL progression and enhance the inhibitory effect of BTK inhibitors(ibrutinib,obrutinib and zbrutinib)on GCB-DLBCL and ABC-DLBCL cells.And in vivo study,Huaier significantly inhibited tumor growth in human GCB-DLBCL and ABC-DLBCL xenograft models.Conclusions: These results indicate that Huaier could inhibit the proliferation and induces apoptosis of GCB-DLBCL cells and ABC-DLBCL cells,and could enhance the inhibitory effect of BTK inhibitors on GCB-DLBCL cells and ABC-DLBCL cells.Our results suggested that Huaier may provide a new strategy for rational drug treatment of DLBCL.更多还原