靶向抑制髓系抑制性细胞调控肿瘤微环境对神经母细胞瘤荷瘤小鼠免疫疗效的影响  

Regulation of tumor microenvironment by targeted inhibiting of myeloid-derived suppressor cells for influencing immune efficacy of neuroblastoma-bearing mice

作　　者：徐伟立[1] 时保军[1] 李索林[1] 于风雪[1] 李萌[1] 郭丽娜[1] 周辉[1] Xu Weili;Shi Baojun;Li Suolin;Yu Fengxue;Li Meng;Guo Lina;Zhou Hui(Department of Pediatric Surgery,Second Hospital,Hebei Medical University,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院小儿外科,石家庄050000

出　　处：《中华小儿外科杂志》2020年第9期846-854,共9页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金面上项目(81472503)。

摘　　要：目的探索靶向抑制髓系抑制性细胞(myeloid-derived suppressor cell,MDSC)调控神经母细胞瘤(neuroblastoma,NB)肿瘤微环境对增强效应细胞体内杀伤效应、提高NB免疫治疗效果的作用和机制。方法以BALB/c小鼠为实验对象,利用NB细胞注射建立荷瘤小鼠模型。按随机数字表法将小鼠随机分为多柔比星(doxorubicin,DOX)2.5 mg/kg组、DOX 5 mg/kg组、多巴胺(dopamine,DA)50 mg/kg组和对照组进行药物筛选,每组20只。分别于NB细胞接种后第7天和第12天注射DOX、DA,于NB细胞接种后第14、17、23天检测比较各组小鼠瘤体内MDSC数量分布及凋亡率、T细胞周期、Treg水平和TAM分布,MDSC培养上清中Arg-1、iNOS、ROS、IL-10、TGF-β含量,比较各组肿瘤生长曲线。根据抑瘤效果选择抑制MDSC最佳药物及浓度。制备NB抗原特异性CTL。按随机数字表法将荷瘤小鼠随机分为DOX组、CTL组、抗GD2组、DOX+CTL组、DOX+抗GD2组和对照组进行免疫治疗对比研究,每组20只。在抑制MDSC基础上实施NB荷瘤小鼠免疫治疗,检测比较各组肿瘤生长曲线,对比各组瘤体中浸润CTL、颗粒酶和穿孔素表达,外周血IL-2和IFN-γ水平。采用SPSS 26.0软件进行统计学处理。结果随时间变化,各药物筛选组和对照组比较,MDSC增长比例显著降低(P=0.001),瘤体中T细胞增殖和对照组比较先升(P<0.001)后抑(P>0.05);TAM由M1型向M2型极化减弱;Treg水平受抑;MDSC培养上清中Arg-1、iNOS、ROS和IL-10含量和对照组比较均降低,且差异均有统计学意义(P均<0.05);不同药物筛选组肿瘤体积比较,差异有统计学意义(P=0.018)。以上各指标均以DOX2.5组表现最为显著,肿瘤生长最缓慢,抑制MDSC作用最强。小剂量DOX干扰后荷瘤小鼠实施免疫治疗,和对照组比较,各组瘤体浸润CD4+、CD8+淋巴细胞比例上升,颗粒酶、穿孔素浓度上升,外周血IL-2、IFN-γ浓度上升,且差异均有统计学意义(P均=0)。以上各指标均以DOX2.5+抗GD2组�Objective To explore the role and mechanism of enhancing the killing potency of effector cells in vivo and enhancing immune efficacy of neuroblastoma(NB)by targeted inhibiting of myeloid-derived suppressor cells(MDSCs)for regulating tumor microenvironment.Methods BALB/c mice were selected as experimental subjects and NB cell injection was employed for establishing a tumor-bearing murine model.The animals were divided randomly into doxorubicin(DOX)2.5 mg/kg,DOX 5 mg/kg,dopamine(DA)50 mg/kg and control groups for drug screening(n=20 each).DOX/DA was injected at 7/12 days post-inoculation.Then tumor indices at 14/17/23 days post-inoculation were recorded and compared,including tumor growth curve,quantity,distribution and apoptotic rate of MDSC,T cell cycle,Treg level and TAM distribution,contents of Arg-1,iNOS,ROS,IL-10 and TGF-βin MDSC culture supernatant.Based upon the anti-tumor effect,optimal drug and its concentration were selected for inhibiting MDSC.Thereafter NB antigen specific CTLs were prepared.For this comparative study of immunotherapy,tumor-bearing mice were randomly divided into DOX,CTL,anti-GD2,DOX+CTL,DOX+anti-GD2 and control groups(n=20 each).Tumor-bearing mice were treated on the basis of MDSC inhibition.Tumor growth curves,infiltrating CTL,perforin and granzyme and levels of IL-2 and IFN-γin peripheral blood were compared between groups.SPSS 26.0 software was employed for statistical processing.Results With the elapsing of time,comparing between drug screening and control groups,the growth rate of MDSC significantly declined(P=0.001)and proliferation of T cells in tumor spiked initially(P<0.001)then decreased(P>0.05).The polarization of TAM from M1 type to M2 type decreased.Treg level was inhibited.As compared with control group,the contents of Arg-1(P=0.001),iNOS(P=0.046),ROS(P=0)and IL-10(P=0)decreased in MDSC supernatant.Comparison of tumor volume between different drug screening groups showed statistically significant differences(P=0.018).The DOX2.5 group showed the most significant variati

关 键 词：神经母细胞瘤 小鼠 免疫治疗 

分 类 号：R739.41[医药卫生—肿瘤]