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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 changes of CD4 + T cell activity in miR-126 knockout (KD) mice and to explore its significance. Methods The splenic CD4 + CD62L + T cells of miR-126KD mice were sorted by magnetic activated cell sorting (MACS). The expression of miR-126 was detected by real-time quantitative PCR. Fluorescence activated cell sorting ) Were used to detect the expression of CD69, CD62L, CD44 and Ki-67 on CD4 + T cell surface. CD4 + T cells were detected by double staining with AnnexinⅤ / PI and flow cytometry The levels of IL-4, IL-10, IL-12, TGF-β and TNF-α in CD4 + T cells were detected by real- TNF-α), γ interferon (IFN-γ) mRNA levels. Results Compared with wild type (WT) mice, the expression of miR-126 in CD4 + T cells in miR-126KD mice was significantly down-regulated. The FACS results showed that the proportion of CD62L in CD4 + T cells in miR-126KD mice Significantly decreased while the percentage of CD69, CD44 and Ki-67 cells was significantly increased, the proportion of CD4 + T cells in vivo significantly decreased; IL-4, IL-10 mRNA levels in CD4 + T cells decreased significantly, -12, TGF-β, TNF-α and IFN-γ mRNA levels were significantly increased. Conclusion The activity of CD4 + T cells in miR-126KD mice significantly increased and promoted their differentiation into Th1 cells.