MicroRNA-146a在脓毒症髓源抑制细胞中的表达及其意义

Expression of microRNA-146a in myeloid derived suppressor cells and its significance

目的比较脓毒症3和7 d的髓源抑制细胞(MDSC)表型差异,了解其与MDSC增殖活性及免疫功能变化间的关系,通过检测MDSC中微小RNA-146a(microRNA-146a,miR-146a)的表达,探讨miR-146a对脓毒症MDSC免疫抑制活性的影响。方法采用盲肠穿孔结扎技术制备C57BL/6J小鼠脓毒症模型,运用磁珠分选方法收集分离CLP后3和7 d时小鼠骨髓MDSC(CD11b+Gr1+细胞),然后进行体外培养并给予脂多糖(LPS)刺激,收集细胞培养上清液,并检测3和7 d时分泌的细胞因子(TNF-α、TGF-β、IL-6、IL-10)和精氨酸酶活性,以及检测对T细胞的抑制作用与miR-146a的表达。结果 CLP后3 d时MDSC以分泌促炎因子(TNF-α、IL-6)为主,CLP后7 d时以分泌抗炎因子(IL-10、TGF-β)为主。CLP后7 d比3 d时表现出更强的T细胞免疫抑制活性。qRT-PCR检测小鼠骨髓MDSC中miR-146a表达,发现CLP 7 d时比3 d时的miR-146a表达量显著升高(P<0.05)。结论在脓毒症的进展过程中,MDSC表型随之发生演变,脓毒症晚期MDSC免疫抑制活性较早期显著增强。miR-146a表达上调可能是脓毒症后期MDSC免疫抑制活性增强的重要机制。

Objective To compare the phenotype of myeloid derived suppressor cells（ MDSCs） separated from the bone marrow of mice 3 d and 7 d after cecal ligation and puncture（ CLP） and to elucidate its potential role in the accumulation and immuno-function of MDSCs by determining the expression of microRNA-146a（ miR-146a） in order to explore the effect of miR-146 a on immonosuppression of MDSCs in sepsis. Methods A septic model was prepareol by CLP in adult male C57 BL /6J mice. MDSCs（ expressing cell-surface CD11 b and GR-1 antigens） from bone marrow were harvested 3 and 7days after CLP and were separated with magnetic bead sorting technique. Then,cytokines secretion and arginase-I activity were detected and the T cell proliferation in vitro and the expression of miR-146 a of MDSCs（ 3 d and 7 d after CLP） were observed. Results MDSCs secreted mostly such promoting inflammatory factors as TNF-α,IL-6 3 days after CLP,but 7days after CLP,they primarily secreted IL-10 and TGF-β which were anti-inflammatory factors. MDSCs had potent immunosuppressive properties by increasing T cell suppression in a late anti-inflammatory phase（ CLP3 d vs CLP7 d,P〈0. 05）. In the meantime,miR-146 a of the MDSCs in bone marrow was overexpressed in septic mice at 7 days（ P〈0. 05）.Moreover,the expression of miR-146 a of the MDSCs in bone marrow of septic mice was higher at 7 days than at 3 days after CLP（ P〈0. 05）. Conclusion The data indicate that the phenotype of MDSCs evolves through early pro-inflammatory phase into the late anti-inflammatory phase. MDSCs have potent immunosuppressive properties in the late phase of sepsis.miR-146 a might play a crucial role in the regulation of immunosuppressive activity of MDSCs in late sepsis.