抗原85B抑制自噬促进霍奇金淋巴瘤小鼠肿瘤凋亡机制研究  

Mechanism of Antigen 85B(Ag85B)inhibition of autophagy promoting tumor apoptosis in Hodgkin lymphoma(HL)mice

作　　者：程永凤 沈亦平[2] 樊春艳 古丽巴哈·买买提[1] 王学梅[1] 李丹露 严媚[1] CHENG Yongfeng;SHEN Yiping;FAN Chunyan;Gulibaha Maimaiti;WANG Xuemei;LI Danlu;YAN Mei(Department of Pediatrics,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Division of Genetics and Genomics,Boston Children's Hospital,Harvard Medical School,Boston 02115,USA)

机构地区：[1]新疆医科大学第一附属医院儿科,乌鲁木齐830054 [2]哈佛大学医学院波士顿儿童医院遗传学与基因组医学,美国波士顿02115

出　　处：《新疆医科大学学报》2025年第3期286-292,298,共8页Journal of Xinjiang Medical University

基　　金：国家自然科学基金项目(8216001,82071276);新疆维吾尔自治区自然科学基金青年科学基金项目(2022D01C739)。

摘　　要：目的研究抗原85B(Antigen 85B,Ag85B)对霍奇金淋巴瘤(Hodgkin lymphoma,HL)小鼠肿瘤生长、凋亡的作用及机制。方法取HDLM-2细胞,用杜氏改良鹰培养基(Dulbecco′s modified eagle medium,DMEM)调整细胞密度为5×10^(6)个/200μL备用。取40只小鼠随机分为4组,小鼠左侧腋下酒精棉片擦拭消毒后给予接种相应试剂。Ⅰ组:接种DMEM基础培养基(200μL);Ⅱ组:接种Ag85B(每克小鼠接种4μg Ag85B,用DMEM基础培养基调整总液量为200μL);Ⅲ组:接种HDLM-2(5×10^(6)个/200μL);Ⅳ组:接种HDLM-2(5×10^(6)个/200μL)+Ag85B;(每克小鼠接种4μg Ag85B,直接混入HDLM-2细胞,维持总液量为200μL)。每日接种一次,连续接种3日。期间每日观察各组小鼠活动、精神状况,毛发和饮食饮水情况。实验第7天、第14天、第21天、第28天、第35天小鼠称重并测量肿瘤的长径及短径,肿瘤体积达到1000 mm3说明建模成功。第35天处死小鼠,剥离瘤体称重,测量肿瘤体积,计算抑瘤率。苏木精-伊红染色(Hematoxylin and Eosin,HE)比较I组、II组小鼠心、肝、脾、肺、肾组织结构。取Ⅲ组、Ⅳ组小鼠肿瘤组织进行后续检测比较,免疫组织化学(Immunohistochemistry,IHC)检测LC3、p62、Beclin-1、Caspase-3、Ki-67在肿瘤组织的定位及蛋白表达;免疫荧光检测Ki-67表达;TUNEL染色检测凋亡;透射电镜(Transmission electron microscopy,TEM)检测自噬体数量;实时荧光定量聚合酶链式反应法(Quantitative real-time polymerase chain reaction,qRT-PCR)、蛋白质印迹法(Western blot)分别检测LC3、p62、Beclin-1、PI3K、AKT、mTOR mRNA和蛋白表达。结果各组小鼠体重随时间延长有下降趋势,各组间比较差异无统计学意义(P>0.05)。Ⅰ组、Ⅱ组小鼠毛发有光泽,精神尚可,活动正常,未见肿瘤生长。Ⅲ组、Ⅳ组小鼠精神萎靡、活动减少、毛发无光泽,有肿瘤生长。与Ⅲ组比较,Ⅳ组小鼠肿瘤体积较小,差异有统计学意义(P<0.05),Ag85B对肿瘤抑制率为Objective To investigate the role and mechanism of Antigen 85B(Ag85B)in tumor growth and apoptosis in Hodgkin lymphoma(HL)mouse models.Methods HDLM-2 cells were resuspended in Dulbecco's modified Eagle medium(DMEM)to a density of 5×10^(6)cells/200μL.40 mice were randomly divided into 4 groups and inoculated as follows:GroupⅠ:DMEM base medium(200μL);GroupⅡ:Ag85B(4μg per gram of mouse,with DMEM to adjust the total volume to 200μL);GroupⅢ:HDLM-2 cells(5×10^(6)cells/200μL);GroupⅥ:HDLM-2 cells(5×10^(6)cells/200μL)+Ag85B,(4μg per gram of mouse,mixed directly with HDLM-2 cells,total volume 200μL).Inoculations were performed daily for 3 days.Mice were monitored daily for activity,mental status,fur condition and food/water intake.On days 7,14,21,28 and 35,mice were weighed and tumor length and width were measured to calculate tumor volume.Tumor growth was considered successful when volume reached 1000 mm3.On day 35,mice were sacrificed,tumors were excised and weighed,and tumor volume was measured to calculate tumor inhibition rate.Hematoxylin and eosin(HE)staining was performed to compare organ histology in GroupsⅠandⅡ.Tumor tissue from GroupsⅢandⅣwas subjected to immunohistochemistry(IHC)for LC3,p62,Beclin-1,Caspase-3 and Ki-67 expression,immunofluorescence for Ki-67,TUNEL staining for apoptosis and transmission electron microscopy(TEM)to assess autophagosome formation.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were used to measure mRNA and protein expression of LC3,p62,Beclin-1,PI3K,AKT and mTOR.Results The body weight of mice in all groups showed a downward trend over time,with no significant differences between the groups(P>0.05).Mice in GroupsⅠandⅡhad glossy fur,were active,and itshowed no tumor growth.Mice in GroupsⅢandⅣwere lethargic,had dull fur and showed tumor growth.Compared to GroupⅢ,GroupⅣhad smaller tumor volumes,with a significant difference(P<0.05),and Ag85B demonstrated a tumor inhibition rate of 20.53%.Hematoxylin and eosin(HE)staining

关 键 词：霍奇金淋巴瘤 抗原85B 自噬 凋亡 增殖 

分 类 号：R733[医药卫生—肿瘤]