Cbl-b 基因沉默对小鼠原代淋巴细胞免疫活性影响的研究  

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

作　　者：胡彬[1] 倪娜娜[1] 吕雅琳[1] 陈浩[1] 刘毅[1] 孙建方[1] 

机构地区：[1]中国医学科学院 北京协和医学院 皮肤病研究所病理科,南京210042

出　　处：《中华皮肤科杂志》2016年第3期168-171,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171513）;江苏省自然科学基金（BK2012506）

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 Entranster^TM-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。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 Entranster^TM-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. Conclusion 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.

关 键 词：黑色素瘤 基因沉默 小鼠 淋巴细胞 RNA 小分子干扰 免疫活性 CBL-B 

分 类 号：R739.5[医药卫生—肿瘤]