南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (07): 800-.

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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

王震，黄文，岑柏宏，魏媛怡，廖路敏，黎国仙，季爱民   

  • 出版日期:2018-07-20 发布日期:2018-07-20

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

  • Online:2018-07-20 Published:2018-07-20

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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-L1 siRNA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-L1 siRNA分子可以用于设计和制备预防、治疗多种癌症的核酸药物。

Abstract: 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.