组织蛋白酶G通过增加胶质瘤干细胞表面MHC-Ⅰ表达提高胶质瘤对T细胞治疗的敏感性  

Cathepsin G increases treatment sensitivity of T cells by enhancing major histocompatibility complex-I special expression on glioma stem cell membrane

作　　者：李锡清[1] 赵尊兰[1] 孔存权[1] 赵黎明[1] 张玉薇 韩双印 Li Xiqing;Zhao Zunlan;Kong Cunquan;Zhao Liming;Zhang Yuwei;Han Shuangyin(Department of Medical Oncology,Henan Provineial People's Hospital,Zhengzhou 450003,China;Center for Translational Medicine,People's Hospital of Zhengzhou University,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院肿瘤内科,郑州450003 [2]郑州大学人民医院干细胞转化医学中心,郑州450003

出　　处：《中华神经医学杂志》2020年第3期217-223,共7页Chinese Journal of Neuromedicine

摘　　要：目的探讨组织蛋白酶G(CatG)提高T细胞治疗胶质瘤效果的机制。方法(1)收集胶质瘤数据库中397例胶质瘤患者的临床资料,采用Kaplan-Meier法进行生存分析,Pearson相关性分析胶质瘤组织中CTSG mRNA与β2微球蛋白(β2M)mRNA表达的相关性。(2)从胶质母细胞瘤中分离培养胶质瘤干细胞(GSC)387和GSC3565,分别分化培养获得胶质瘤分化细胞(DGC)387和DGC3565。取GSC387细胞,分为CatG组、CatG抑制物组,分别用0.1μg/μL重组人类白细胞抗原(HLA)-A^*02:01和HLA-B^*15:01与4 ng/μL CatG、1O μmol/L CatG抑制物共培养10 min,应用托马斯亮蓝染色检测细胞HLA-A^*02:01和HLA-B^*15:01的表达,应用Western blotting检测细胞主要组织相容性复合体(MHC)-I、MHC-DR蛋白的表达。(3)取GSC387、GSC3565细胞和DGC387、DGC3565细胞,分别分为4组(CatG组、CatG抑制组、空白抗体1组,空白抗体2组),分别加人4 ng/μL CatG,10μmol/L CatG抑制物、空白抗体1、空白抗体2处理24 h,采用流式细胞仪检测细胞HLA-ABC的表达。⑷取GSC387、GSC3565细胞和DGC387、DGC3565细胞,分别分为CatG组和CatG抑制组,釆用荧光素酶实验检测CatG对T、自然杀伤细胞杀伤作用的影响。结果(1)CTSG mRNA高表达组患者的生存率高于CTSG mRNA低表达组,β2M mRNA低表达组患者的生存率高于β2M mRNA高表达组,差异均有统计学意义(P<0.05)。相关性分析显示胶质瘤组织中CTSG mRNA与β2MmRNA的表达呈负相关关系(r=-9.160,P=0.000),(2)托马斯亮蓝染色检测显示:与CatG抑制物组比较,CatG组细胞HLA-A^*02:01和HLA-B^*15:01的表达增加。Western blotting检测显示:与CatG抑制物组比较,CatG组细胞MHC-I的表达增加,MHC-DR的α和β链降低。(3)流式细胞仪检测显示:CatG组GSC387、GSC3565细胞HLA-ABC的表达高于CatG抑制物组,差异有统计学意义(P<0.05)。(4)荧光素酶实验显示:与CatG抑制物组比较,CatG组T细胞对GSC细胞的杀伤比例增加,差异有统计学意义(P<0.05)。结论CatG可�Objective To investigate the mechanism of cathepsin G(CatG)in improving the treatment efficacy of T cells in gliomas.Methods(1)Clinical data of 397 glioma patients in the glioma database were collected,Kaplan-Meier method was used to perform survival analysis,and the correlation between CTSG and β2-microglobulin(β2M)mRNA expressions in glioma tissues was analyzed.(2)Glioma stem cell(GSC)387 and GSC3565 were isolated from glioblastoma and differentiated into differentiated glioma cell(DGC)387 and DGC3565,respectively;GSC387 was divided into CatG group and CatG inhibitor group,and cells in the CatG group and CatG inhibitor group were cultured with 0.1μg/μL recombinant human leukocyte antigen(HLA)-A^*02:01 and HLA-B^*15:01 combined with 4 ng/μL CatG or 10 mol/L CatG inhibitor for 10 min,respectively;the expressions of HLA-A^*02:01 and HLA-B^*15:01 were detected by Thomas bright blue staining,and the protein expressions of major histocompatibility complex(MHC)-I and MHC-DR were detected by Western blotting.(3)GSC387,GSC3565,DGC387,and DGC3565 were divided into 4 groups,including CatG group,CatG inhib让ory group,blank antibody group 1 and blank antibody group 2,respectively;4 ng/μL CatG,10 μmoI/L CatG inhibitor,blank antibody 1 and blank antibody 2 were added into the cells from the 4 groups for 24 h,and the expression of HLA-ABC was detected by flow cytometry.(4)GSC387,GSC3565,DGC387,and DGC3565 were divided into CatG group and CatG inhibitory group,respectively;luciferase assay was used to detect the influence of CatG in the killing effects of T cells and natural killer cells.Results(1)The survival rate in patients from CTSG mRNA high expression group was significantly higher than that in patients from CTSG mRNA low expression group,and the survival rate in patients from β2M mRNA low expression group was statistically higher than that in patients from β2M mRNA high expression group(P<0.05);a negative correlation between CTSG mRNA and β2M mRNA expressions was noted in glioma tissues(r=-9.160,P=0.000).(2)Th

关 键 词：神经胶质瘤 组织蛋白酶G 免疫治疗 CTSG基因 

分 类 号：R73[医药卫生—肿瘤]