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目的观察外源性一氧化碳释放分子3(carbon monoxide-releasing molecule 3,CORM-3)对肾固有树突状细胞(renal dendritic cells,rDCs)活化的影响,并探讨其可能的作用机制。方法选取C57BL/6J(H-2kb)小鼠制备肾脏单细胞悬液,采用磁珠分选CD11c+rDCs,并用流式细胞术鉴定rDCs纯度。用CORM-3或无活性的CORM(inactive CO-releasing mol-ecule,iCORM)处理新鲜肾脏分离的rDCs,实时定量RT-PCR检测TLR4基因表达,ELISA法检测rDCs培养液上清中TNF-α蛋白水平。建立TLR4-/-(C3H/HeJ)和TLR4+/+(C3H/HeN)小鼠肾脏热缺血30 min+冷保存24 h模型,在冷保存期间经CORM-3处理之后分选rDCs,抽提RNA,行实时定量RT-PCR检测TNF-α基因表达。结果 CORM-3呈剂量依赖性地抑制小鼠未成熟rDCs的TLR4 mRNA表达(P<0.05)。与iCORM比较,CORM-3能够抑制LPS刺激后的rDCs表达TNF-α(P<0.01)。当TLR4缺陷之后,CORM-3不再抑制rDCs表达TNF-α。结论 CORM-3可显著抑制肾脏未成熟rDCs表达TLR4,也可抑制LPS刺激和缺血损伤时炎症因子的表达,但对于TLR4缺陷小鼠这一抑制作用消失,提示CORM-3对内源性配体介导的rDCs活化过程的抑制作用由TLR4信号通路介导。
Objective To investigate the effect of exogenous carbon monoxide-releasing molecule 3 (CORM-3) on the activation of renal dendritic cells (rDCs) and to explore its possible mechanism. Methods C57BL / 6J (H-2kb) mice were used to prepare single cell suspension of kidney. CD11c + rDCs were sorted by magnetic beads. The purity of rDCs was identified by flow cytometry. The rDCs isolated from fresh kidneys were treated with CORM-3 or inactive CO-releasing mol-ecule (iCORM). The expression of TLR4 gene was detected by real-time quantitative RT-PCR. The TNF- Level. The renal ischemia of TLR4 - / - (C3H / HeJ) and TLR4 + / + (C3H / HeN) mice was established for 30 min and stored for 24 h in cold storage. After being treated with CORM-3 during cold storage, rDCs were sorted and RNA was extracted , Real-time quantitative RT-PCR detection of TNF-α gene expression. Results CORM-3 inhibited the TLR4 mRNA expression in immature rDCs in a dose-dependent manner (P <0.05). Compared with iCORM, CORM-3 could inhibit the expression of TNF-α in rDCs stimulated by LPS (P <0.01). CORM-3 no longer inhibits rDCs from expressing TNF-α after TLR4 deficiency. Conclusions CORM-3 can significantly inhibit the expression of TLR4 in immature rDCs of kidney and the expression of inflammatory cytokines in LPS-stimulated and ischemic injury, but disappeared in TLR4-deficient mice, suggesting that CORM-3 inhibits the endogenous The inhibition of body-mediated activation of rDCs is mediated by the TLR4 signaling pathway.