中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (3): 168-171.

• 论著 • 上一篇    下一篇

Cbl-b基因沉默对小鼠原代淋巴细胞免疫活性影响的研究

胡彬1,2,倪娜娜2,吕雅琳3,陈浩4,刘毅3,孙建方4   

  1. 1. 武汉市第一医院
    2. 中国医学科学院皮肤病研究所
    3. 中国医学科学院北京协和医学院皮肤病研究所
    4. 南京 中国医学科学院北京协和医学院皮肤病研究所
  • 收稿日期:2015-06-08 修回日期:2015-11-07 出版日期:2016-03-15 发布日期:2017-03-20
  • 通讯作者: 孙建方 E-mail:fangmin5758@aliyun.com
  • 基金资助:
    泛素连接酶Cbl-b基因沉默联合PD-1通路阻断对小鼠恶性黑素瘤TRP-2肽疫苗治疗效应的影响及机制研究

In vitro effects of Cbl-b gene silencing on immunocompetence of primary murine lymphocytes

  • Received:2015-06-08 Revised:2015-11-07 Online:2016-03-15 Published:2017-03-20

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摘要： 目的 体外初步研究Cbl-b基因经特异性siRNA沉默后对小鼠淋巴细胞免疫活性的影响。 方法 摘取C57BL/6小鼠的脾脏，体外无菌分离小鼠脾脏淋巴细胞后进行培养。通过EntransterTM-R4000试剂将Cbl-b siRNA转染入小鼠原代淋巴细胞以沉默细胞内Cbl-b的表达。转染72 h后通过酶联免疫吸附实验（ELISA）检测淋巴细胞培养上清中干扰素γ（INF-γ）及肿瘤坏死因子α（TNF-α）的表达量。通过与B16F10黑素瘤细胞共培养，研究Cbl-b基因沉默后的淋巴细胞对B16F10黑素瘤细胞免疫杀伤活性的影响。 结果 Cbl-b siRNA成功转染进小鼠原代淋巴细胞并能有效沉默细胞内Cbl-b的表达。与阴性对照转染组及空白组相比，转染Cbl-b siRNA的淋巴细胞IFN-γ、TNF-α分泌量增加（P < 0.05）。共培养检测结果显示，Cbl-b siRNA转染组比转染阴性对照组能更高效地杀伤小鼠B16F10细胞。 结论 Cbl-b基因沉默能够促进小鼠淋巴细胞INF-γ、TNF-α分泌能力，并能增强淋巴细胞对B16F10黑素瘤细胞的体外免疫杀伤作用。

Abstract: Hu Bin, Ni Nana, Lyu Yalin, Chen Hao, Liu Yi, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （the current affiliation of the first author was Department of Dermatology, Wuhan No. 1 Hospital, Wuhan 430222, China） Corresponding authors: Sun Jianfang, Email: Sunjf57@163.com; Liu Yi, Email: dr.liuyi@gmail.com 【Abstract】 Objective To evaluate in vitro effects of specific small interfering RNA （siRNA）-silencing of the casitas B-lineage lymphoma b （Cbl-b） gene on immunocompetence of primary murine lymphocytes. Methods Spleens were resected from C57BL/6 mice, and splenic lymphocytes were sterily isolated and cultured in vitro. These lymphocytes were divided into 3 groups: silence group transfected with a Cbl-b-specific siRNA using the EntransterTM-R 4000 reagent, negative control group transfected with a negative control siRNA using the EntransterTM-R4000 reagent, blank control group receiving no treatment. After additional culture for 72 hours, ELISA was performed to measure levels of interferon γ （IFN-γ） and tumor necrosis factor α （TNF-α） in culture supernatants of lymphocytes. In addition, the Cbl-b gene-silenced lymphocytes were co-cultured with B16F10 melanoma cells to evaluate their immunocytotoxic effects on melanoma cells. Results Splenic lymphocytes were successfully isolated from C57BL/6 mice and cultured in vitro, and the Cbl-b-specific siRNA was also successfully transfected into the primary murine lymphocytes and effectively down-regulated the expression of Cbl-b gene in them. Compared with the negative control group and blank control group, the silence group showed significantly increased supernatant levels of IFN-γ and TNF-α （all P < 0.05）. The immunocytotoxic effect of lymphocytes on melanoma cells was significantly stronger in the silence group than in the negative control group. Conclusions Cbl-b gene silencing can promote secretion of IFN-γ and TNF-α by murine lymphocytes, and enhance their immunocytotoxic effects on B16F10 melanoma cells in vitro.