[1]陈赛明,李智群,周利民,等.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[J].南方医科大学学报,2019,(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()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2019年05期
页码:
554
栏目:
出版日期:
2019-05-15

文章信息/Info

Title:
Cbl-b gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells
作者:
陈赛明李智群周利民张云霞
关键词:
基因沉默泛素连接酶人喉鳞癌免疫杀伤
Keywords:
gene silencing ubiquitin ligase human laryngeal squamous cell carcinoma immune killing
DOI:
10.12122/j.issn.1673-4254.2019.05.09
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.

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更新日期/Last Update: 1900-01-01