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目的研究微小RNA-126(microRNA-126,miR-126)基因敲减(knock down,KD)后对CD4~+T细胞体外功能的影响并探讨其意义。方法 MACS分选miR-126 KD小鼠脾脏CD4~+CD62L~+T细胞,ConA刺激48h后,Real-time PCR技术检测细胞内miR-126成熟体表达水平;FACS分别检测CD4~+T细胞表面活化分子CD69、CD62L、CD44和增殖相关分子Ki-67,以及细胞因子IFN-γ、IL-4、IL-17表达变化;Annexin V/PI双染色法检测CD4~+T细胞的凋亡情况。结果与野生型(wide-type,WT)小鼠相比,miR-126KD小鼠的CD4~+T细胞中miR-126成熟体表达显著下调(P<0.001);miR-126 KD小鼠CD4~+T细胞活化后CD62L的表达比例显著减少(P<0.01),而CD69、CD44的比例显著增加(P<0.05);同时Ki-67~+的比例也明显增加(P<0.05);此外,miR-126 KD小鼠CD4~+T细胞的INF-γ表达比例显著上调(P<0.001),IL-4的比例显著下调(P<0.01),而IL-17的比例没有明显变化(P>0.05);最后,CD4~+T细胞的凋亡比例显著减少(P<0.05)。结论 miR-126基因敲减后CD4~+T细胞的活化、增殖和相关细胞因子分泌均明显增强,为后续深入探讨miR-126在免疫应答中的作用及机制提供前期实验基础。
Objective To investigate the effect of microRNA-126 (miR-126) knockdown (KD) on the function of CD4 ~ + T cells in vitro and its significance. Methods The splenic CD4 ~ + CD62L ~ + T cells of miR-126 KD mice were isolated by MACS and the expression of miR-126 was detected by Real-time PCR 48h after ConA stimulation. The surface of CD4 ~ + T cells were detected by FACS, The expressions of CD69, CD62L, CD44, Ki-67 and IFN-γ, IL-4 and IL-17 were detected by flow cytometry. The apoptosis of CD4 ~ + T cells was detected by Annexin V / PI double staining. Results Compared with wild-type (WT) mice, miR-126 mature CD4 + T cells in miR-126KD mice were significantly down-regulated (P <0.001) (P <0.01), while the ratio of CD69 and CD44 increased significantly (P <0.05), while the proportion of Ki-67 ~ + also increased significantly (P <0.05). In addition, The proportion of IL-4 in CD4 ~ + T cells in miR-126 KD mice was significantly increased (P <0.001), the proportion of IL-4 was significantly decreased (P <0.01) 0.05). Finally, the percentage of apoptosis of CD4 ~ + T cells was significantly decreased (P <0.05). Conclusion The activation and proliferation of CD4 ~ + T cells after knockdown of miR-126 gene and the secretion of related cytokines were significantly increased, providing the experimental basis for further study on the role and mechanism of miR-126 in immune response.