摘要
目的观察miR-126基因敲减(KD)小鼠体内CD4+T细胞活性的变化并探讨其意义。方法利用磁珠活化细胞分选技术(MACS)分选miR-126KD小鼠脾脏CD4+CD62L+T细胞,实时定量PCR检测细胞内miR-126成熟体的表达水平;荧光激活细胞分选术(FACS)检测CD4+T细胞表面活化标志CD69、CD62L和CD44分子以及Ki-67的表达变化;膜联素Ⅴ/碘化丙啶(annexinⅤ/PI)双染色法结合流式细胞术检测CD4+T细胞的凋亡情况;实时定量PCR检测CD4+T细胞功能相关细胞因子白细胞介素4(IL-4)、IL-10、IL-12、转化生长因子β(TGF-β)、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的mRNA水平变化。结果与野生型(WT)小鼠组相比,miR-126KD小鼠组CD4+T细胞内miR-126成熟体的表达显著下调;FACS结果显示miR-126KD小鼠CD4+T细胞表达CD62L的比例明显减少,而表达CD69、CD44以及Ki-67细胞的比例显著增加,CD4+T细胞的体内凋亡比例显著减少;CD4+T细胞中IL-4、IL-10的mRNA水平明显降低,而IL-12、TGF-β、TNF-α以及IFN-γ的mRNA水平显著增加。结论 miR-126KD小鼠体内CD4+T细胞的活性显著增强并促进其向Th1细胞分化。
Objective To investigate the change of CD4 ＋ T cell activity in microRNA-126（ miR-126） knockdown（ KD）mice and explore its significance. Methods The expression level of mature miR-126 in CD4＋CD62L＋T cells purified by magnetic-activated cell sorting（ MACS） was analyzed by real-time PCR using specific probe. Furthermore,the expression levels of CD69,CD62 L and CD44 molecules,as well as intracellular proliferating nuclear antigen Ki-67,in CD4＋T cells in miR-126 KD mice were detected by fluorescence-activated cell sorting（ FACS）. Moreover,the apoptosis of CD4＋T cells was analyzed by annexin Ⅴ / PI staining assay combined with flow cytometry. Finally,the relative expressions of function-related cytokines including interleukine 4（ IL-4）,IL-10,IL-12,transforming growth factor（ TGF-β）,interferon（ IFN-γ） and tumor necrosis factor（ TNF-α） in CD4＋T cells were determined by real-time PCR. Results Compared with wild-type（ WT） mice,the expression level of mature miR-126 in CD4＋T cells in miR-126 KD mice was dramatically reduced. Furthermore,the proportion of CD62L＋in CD4＋T cells also decreased significantly,while the proportions of CD69＋,CD44＋and Ki-67＋cells were remarkably elevated. Meanwhile,the apoptosis proportion of CD4＋T cells in vivo dropped dramatically in miR-126 KD mice. Finally,the mRNA expressions of IL-4 and IL-10 in CD4＋T cells were significantly downregulated,but IL-12,TGF-β,TNF-α and IFN-γ mRNAs were obviously up-regulated. Conclusion miR-126 knockdown could significantly enhance the functional activity of CD4＋T cells in vivo and promote cell differentiation into Th1 cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第3期347-351,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31370918)
教育部新世纪优秀人才计划(NCET-12-0661)
