南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (05): 554-.doi: 10.12122/j.issn.1673-4254.2019.05.09

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Cbl-b基因沉默增强T细胞对人喉鳞癌细胞Hep-2 的免疫杀伤效果

陈赛明，李智群，周利民，张云霞   

  • 出版日期:2019-05-20 发布日期:2019-05-20

Cbl-b gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells

  • Online:2019-05-20 Published:2019-05-20

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摘要： 目的研究泛素连接酶（Cbl-b）基因沉默对H9 人T淋巴细胞免疫杀伤人喉鳞癌细胞Hep-2 作用的影响和相关机制。方 法选择12例喉鳞癌患和12例健康对照者，用磁性细胞分离技术分离外周血CD4+T细胞应用RT-PCR法检测Cbl-b的表达水 平。同时，将人T淋巴细胞H9 接种培养于96 孔板，分为4 组：以脂质体转染法将Cbl-b-siRNA 重组序列转染人T淋巴细胞 H9，作为Cbl-b-siRNA组；Cbl-b-siRNA重组序列转染T细胞成功后，在其培养液中加入IL-2 中和抗体，作为anti-IL-2+Cbl-bsiRNA 组；以脂质体转染法将随机阴性对照序列转染的人T淋巴细胞H9 作为阴性组（NC组）；以不做任何处理人T淋巴细 胞H9 为空白组（BC组）。采用倒置荧光显微镜观察人T淋巴细胞H9 的转染效率、以实时荧光定量PCR法和蛋白免疫印迹 法检测各组人T 淋巴细胞H9 中Cbl-b mRNA和蛋白表达情况。以细胞计数试剂盒（CCK-8）检测各组人T 淋巴细胞H9 对 Hep-2细胞的杀伤作用；流式细胞仪检测各组人T淋巴细胞H9表面活化分子CD69、CD25阳性表达率；采用ELISA法检测T细 胞上清液中白介素-2（IL-2）、γ干扰素（INF-γ）水平。结果Cbl-b mRNA在喉鳞癌患者CD4+T细胞中表达升高，与健康对照差异 有显著性（P<0.05）。Cbl-b-siRNA组和阴性组的转染效率分别为（78.30±6.06）%、（77.25±6.38）%，转染效果良好；Cbl-b-siRNA 组人T淋巴细胞H9 的Cbl-b mRNA和蛋白相对表达量与anti-IL-2+Cbl-b-siRNA组无显著差异（P>0.05），但显著低于空白组 和阴性组（P<0.05）；不同效靶比时，Cbl-b-siRNA组Hep-2 肿瘤细胞杀伤率均显著高于anti-IL-2+Cbl-b-siRNA组、阴性组和空 白组（P<0.05）。Cbl-b-siRNA 组人T淋巴细胞H9 表面活化分子CD69、CD25 阳性表达率均显著高于anti-IL-2+Cbl-b-siRNA 组、空白组和阴性组（P<0.05）；Cbl-b-siRNA 组人T 淋巴细胞H9 上清液中INF-γ、IL-2 水平均显著高于空白组和阴性组（P< 0.05）；空白组与阴性组人T淋巴细胞H9 的Cbl-b mRNA和蛋白相对表达量、肿瘤细胞杀伤率、表面活化分子CD69、CD25 阳 性表达率、上清液中IL-2、INF-γ水平比较差异均不显著（P>0.05）。结论应用siRNA技术沉默T细胞Cbl-b 基因可有效增强 人T淋巴细胞H9 对人喉鳞癌细胞株Hep-2 体外免疫杀伤作用，其机制可能与T细胞Cbl-b 基因沉默促进人T淋巴细胞H9 分 泌IL-2、加强T淋巴细胞激活有关。

Abstract: Objective To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism. Methods CD4+ T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA. Results The CD4 + T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals (P<0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein (P<0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (P>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment (P<0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups (P<0.05). Conclusion Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells in vitro probably as the result of enhanced IL-2 secretion and T lymphocyte activation.