IL-12基因修饰DC中SOCS1基因沉默对体外诱导特异性CTL的影响  

Silencing of SOCS1 in IL-12-transfected DC enhances the power of DC to induce efficacious CTL against lung cancer cells in vitrro

作　　者：田晓静[1] 史兵伟[1] 韩金玲[1] 金文涛[1] 钱高潮[1] 

机构地区：[1]南京中医药大学附属常州市中医院检验科,常州213003

出　　处：《现代免疫学》2015年第5期388-392,共5页Current Immunology

基　　金：国家自然科学基金(81372454)

摘　　要：利用重组腺病毒载体pAd CMV/V5-DEST-IL-12转染小鼠骨髓来源的树突状细胞(IL-12/DC),探讨SOCS1基因沉默IL-12/DC在体外诱导细胞毒性T淋巴细胞(CTL)的效能,及其免疫杀伤肺癌细胞株LLC的能力。采用重组腺病毒介导IL-12基因和SOCS1SiRNA基因共同修饰C57BL/6小鼠骨髓来源的DC,经反复冻融法提取LLC抗原,致敏基因修饰的DC;用ELISA法检测各组DC分泌IL-12和IL-10的水平,及各组DC刺激后的T细胞分泌IFN-γ的水平;MTT法检测DC刺激同源小鼠T细胞的增殖能力,微量细胞毒法检测CTL的活性并收集刺激后的T细胞,流式细胞术分析CD8+/CD4+比例和CD4+CD25+Treg的水平;统计学分析各组间的差异。SOCS1SiRNA和IL-12基因共同修饰能有效下调DC中SOCS1蛋白的表达并上调IL-12蛋白的表达;IL-12的分泌水平也明显高于SOCS1SiRNA或IL-12单基因转染组;基因共同修饰的DC表型更加成熟,能明显促进CTL的增殖和活化,减少Treg的生成;CTL分泌高水平的IFN-γ,产生对LLC特异性的细胞免疫。IL-12 gene and SOCS1SiRNA were simultaneously transfererd into murine bone marrow derived dendritic ceils and the effect of silencing of SOCS1 on the DC with respect to its efficiency of inducing specific CTL were investigated. The SOCS1 and IL-12 expression levels at protein were determined by Western blot; The IL-12 and IL-10 in DC culture supernatant and IFN-γ secreted by CTL were detected by ELISA. The modified DC was loaded with lung cancer antigen LLC. The ability of DCs to stimulate the proliferation of homologous T cells was investigated by MTT assay. Flow cytometry was performed to e valuate the ratio of CD8^＋/CD4^＋ and CD4^＋CD25^＋Treg. CTL killing activity was carried out by standard microcytotoxicity assay. The results showed that adenovirus vector pad CMV/V5-DEST- IL-12 and SOCS1SiRNA gene-modified DCs were constructed successfully. Compared with the single IL-2 or SOCS1SiRNA transfected DC, the co-transferred DS expressed higher levels of IL-12 and lower levels of SOCS1. Besides, the phenotype suggested that the DCs were more mature, and production of IFN-T was elevated. They markedly enhanced the proliferation and cytotoxic activity of CTL and reduced the production of Treg. In conclusion, The gene modified DC loaded with lung carcinoma antigen LLC could obviously activate proliferation of the homologous T cells, and induces CTL killing LLC lung cancer cells by secreting IFN-γ.

关 键 词：白介素-12 SOCS1 树突状细胞 T细胞 肺癌 

分 类 号：R392.12[医药卫生—免疫学]