MYCN通过抑制NK细胞介导神经母细胞瘤细胞免疫逃逸机制研究  

Mechanism study of MYCN mediated immune escape of neuroblastoma cells through inhibiting NK cells

作　　者：吴旻 王军[2] 郭夏 姚聪 蔡秦真 袁文 刘鑫 袁纯辉 彭飞 Wu Min;Wang Jun;Guo Xia;Yao Cong;Cai Qinzhen;Yuan Wen;Liu Xin;Yuan Chunhui;Peng Fei(Department of Hematological Oncology,Municipal Children's Hospital(Wuhan Maternal&Children's Healthcare Hospital),Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430016,China;Department of Laboratory Medicine,Municipal Children's Hospital(Wuhan Maternal&Children's Healthcare Hospital),Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430016,China;Department of Pediatric Healthcare,Municipal Children's Hospital(Wuhan Maternal&Children's Healthcare Hospital),Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430016,China;Department of General Surgery,Municipal Children's Hospital(Wuhan Maternal&Children's Healthcare Hospital),Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430016,China)

机构地区：[1]华中科技大学同济医学院附属武汉儿童医院血液科,武汉430016 [2]华中科技大学同济医学院附属武汉儿童医院检验科,武汉430016 [3]华中科技大学同济医学院附属儿童医院儿童保健部,武汉430016 [4]华中科技大学同济医学院附属儿童医院普外科,武汉430016

出　　处：《中华小儿外科杂志》2022年第10期874-883,共10页Chinese Journal of Pediatric Surgery

基　　金：武汉市卫健委科研项目资助课题(WX20D16,WX20C03,WX18Q03)。

摘　　要：目的探讨MYCN对NK细胞的调控作用及其介导神经母细胞瘤(neuroblastoma,NB)细胞免疫逃逸机制。方法采用荧光原位杂交法(FISH)法和免疫组织化学法检测NB组织MYCN的表达情况,流式细胞术检测外周血NK细胞数量及比例、NK细胞的活化、NK细胞表面活化受体和NB细胞表面相应配体的表达状况;干扰/过表达NB细胞系MYCN,与分选的健康儿童NK细胞进行共培养,ELISA法检测共培养上清中细胞因子的分泌状况,4 h乳酸脱氢酶(LDH)释放法检测NK细胞对NB细胞的杀伤性。结果MYCN高表达NB患儿外周血NK细胞数量(F=45.53,P=0.0002)及比例(F=43.93,P=0.0003)显著减少,MYCN可显著抑制γ-干扰素、颗粒酶B和穿孔素的释放[SK-N-BE(2):t=9.10,P=0.0008;t=4.76,P=0.0089;t=4.66,P=0.0096;SH-SY-5Y:t=16.79,P<0.0001;t=35.85,P<0.0001;t=24.66,P<0.0001],抑制NK细胞的活化和对靶细胞的杀伤作用[SK-N-BE(2):t=7.25,P=0.0019;SH-SY-5Y:t=33.07,P<0.0001],MYCN高表达促进NK细胞NKG2A的表达[SK-N-BE(2):t=9.25,P=0.0008;SH-SY-5Y:t=14.65,P=0.0001],抑制NKG2D的表达[SK-N-BE(2):t=7.21,P=0.0020;SH-SY-5Y:t=8.66,P=0.0010],同时抑制NB细胞中NKG2D配体MICA[SK-N-BE(2):t=18.35,P<0.0001;SH-SY-5Y:t=56.90,P<0.0001]和MICB[SK-N-BE(2):t=22.40,P<0.0001]的表达。结论NB中高表达的MYCN可通过抑制NK细胞的数量及活性从而抑制NK细胞对NB细胞的杀伤介导NB细胞免疫逃逸。Objective To explore the regulatory effect of MYCN on NK cells and to elucidate its mechanism of mediating neuroblastoma(NB)cell immune escape.Methods Fluorescent in situ hybridization(FISH)and immunohistochemistry were utilized for detecting the expression of MYCN in NB tissues.Flow cytometry was employed for detecting the number and proportion of NK cells in peripheral blood,the activation of NK cells,the expression of NK cell surface receptor and corresponding ligand on NB cell surface.NB cell lines were interfered/overexpressed MYCN and co-cultured with NK cells from healthy children.Cytokine secretion in co-cultured supernatant was detected by enzyme-linked immunosorbent assay(ELISA).The destruction of NK cells to NB cells was assayed by 4-h lactate dehydrogenase(LDH)release.Results The number(F=45.53,P=0.0002)and proportion(F=43.93,P=0.0003)of NK cells significantly declined in peripheral blood of NB children with high MYCN expression.MYCN could significantly restrain the release of gamma interferon,granzyme B and perforin[SK-N-BE(2):t=9.10,P=0.0008;t=4.76,P=0.0089;t=4.66,P=0.0096;SH-SY-5Y:t=16.79,P<0.0001;t=35.85,P<0.0001;t=24.66,P<0.0001]and suppress the activation of NK cells[SK-N-BE(2):t=7.25,P=0.0019;SH-SY-5Y:t=33.07,P<0.0001]and killing of target cells.High expression of MYCN promoted the expression of NKG2A[SK-N-BE(2):t=9.25,P=0.0008;SH-SY-5Y:t=14.65,P=0.0001]and arrested the expression of NKG2D[SK-N-BE(2):t=7.21,P=0.0020;SH-SY-5Y:t=8.66,P=0.0010]in NK cells.MYCN over-expression suppressed the expression of NKG2D ligand MICA[SK-N-BE(2):t=18.35,P<0.0001;SH-SY-5Y:t=56.90,P<0.0001]and MICB[SK-N-BE(2):t=22.40,P<0.0001]in NB cells.Conclusions High expression of MYCN in NB may suppress NK cell killing to NB cell and mediate NB cell immune escape through inhibiting NK cell number and activity.

关 键 词：神经母细胞瘤 MYCN基因 自然杀伤细胞 自然杀伤细胞2族成员D 免疫逃逸 

分 类 号：R739.4[医药卫生—肿瘤]