siRNA介导的PD-L1沉默可增强人CD8~＋T淋巴细胞的体外杀伤作用  

Small interfering RNA-mediated programmed cell death-ligand 1 silencing in human glioma cells enhances human CD8^+T lymphocyte cytotoxicity in vitro

作　　者：王震[1,2,3] 黄文 岑柏宏 魏媛怡[1] 廖路敏 黎国仙 季爱民 WANG Zhen;HUANG Wen;CEN Bohong;WEI Yuanyi;LIAO Lumin;LI Guoxian;JI Aimin(Department of Pharmacy,Zhujiang Hospital of Southern Medical University,Guangzhou 510282,China;R＆D Center,Nanjing Pharmaceutical Factory Co.,Ltd.,Nanjing 210007,China;Department of Radiation Oncology,Affiliated Cancer Hospital of Guangzhou Medical University,Guangzhou 510282,China;Department of Pharmacy,Shaoyang Central Hospital,Shaoyang 422000,China)

机构地区：[1]南方医科大学珠江医院药剂科,广东广州510282 [2]南京制药厂有限公司新药研发中心,江苏南京210007 [3]邵阳市中心医院药剂科,湖南邵阳422000 [4]广州医科大学附属肿瘤医院肿瘤放射中心,广东广州510095

出　　处：《南方医科大学学报》2018年第7期800-806,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81370449); 广州市科技计划项目产学研协同创新重大专项(201504291720212); 广东省省级科技计划项目(2013B091300014)。

摘　　要：目的设计并筛选多条可高效特异性降低肿瘤细胞表面沉默程序性死亡受体-配体1(PD-L1)表达的小干扰RNA(siRNA)序列,并研究其增强人CD8^+T淋巴细胞对人胶质瘤细胞(U87 MG)免疫杀伤作用。方法根据siRNA设计法则,并通过siDirect软件在线设计针对PD-L1基因的几种不同的siRNA序列,并运用BLAST进行同源性分析,最终确定候选序列;通过RT-qPCR、Western blot及流式细胞术实验,选出高效抑制PD-L1 mRNA及蛋白表达的siRNA序列;通过流式细胞术及CCK8检测PD-L1siRNA沉默U87 MG细胞的PD-L1后,人CD8^+T细胞对U87 MG的细胞凋亡及增殖的影响。结果设计了10条针对人PD-L1基因具有不同核苷酸序列的siRNA。采用RT-qPCR、Western blot及流式细胞术在mRNA及蛋白水平上检测siRNA的沉默效率,与对照组相比,siPD-L1-1、siPD-L1-2、siPD-L1-3、siPD-L1-4、siPD-L1-5及siPD-L1-8均显著降低U87 MG中PD-L1基因的表达(P<0.05),其中siPD-L1-3的沉默效率最高。与对照组相比,流式细胞术及CCK8结果显示,siPD-L1-3及siPD-L1-8均可显著增强人CD8^+T细胞对U87 MG细胞的杀伤作用,且该杀伤作用可显著抑制U87 MG细胞增殖(P<0.05)。结论本文设计并筛选了能有效沉默PD-L1基因表达的siPD-L1-1、siPD-L1-2、siPD-L1-3、siPD-L1-4、siPD-L1-5及siPD-L1-8的6条序列,其中siPD-L1-3和siPD-L1-8可高效增强T淋巴细胞对U87 MG细胞免疫杀伤作用,且siPD-L1-3的作用最为显著。本文设计的PD-L1siRNA分子可以用于设计和制备预防、治疗多种癌症的核酸药物。Objective To investigate the effect of small interfering RNA（siRNA）-mediated silencing of programmed cell deathligand 1（PD-L1） in human glioma cells on the cytotoxicity of human CD8＋T lymphocytes against the modified tumor cells.Methods A siRNA sequence targeting PD-L1 gene was designed and transfected into human glioma U87 MG cells via lipofectamine 2000, and the gene silencing effect was validated using RT-qPCR, Western blotting, and flow cytometry. The transfected cells were co-cultured with human CD8＋T lymphocytes, and the apoptosis of the tumor cells was analyzed with flow cytometry. Results The siRNA sequence showed strong PD-L1 gene-silencing effect at both mRNA and protein levels in U87 MG cells. Compared with the control cells, the transfected U87 MG cells showed significantly increased vulnerability to the cytotoxicity of human CD8＋T cells and an obvious reduction of proliferative activity in the co-culture（P〈0.05）. Conclusion Transfection of human glioma U87 MG cells with the specific siRNA targeting PD-L1 obviously enhances the toxicity of human T lymphocytes in the co-culture.

关 键 词：程序性死亡受体-配体1 小干扰RNA 神经胶质瘤细胞 CD8+T细胞 核酸药物 

分 类 号：R730.5[医药卫生—肿瘤]