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目的探讨以白细胞介素-1受体相关激酶-4(IRAK-4)特异性短发夹RNA(shRNA)阻断内毒素诱导的库普弗细胞(KCs)激活效应的可行性。方法构建两对表达IRAK-4-shRNA的阳性载体质粒(pSⅡRAK-4-A,pSⅡRAK-4-B)及一对阴性载体质粒(pSⅡRAK-4-C)。分离培养小鼠KCs,分为正常对照组,RNA干扰(RNAi)对照组(转染pSⅡRAK一4 C)与RNAi抑制组(转染pSⅡRAK-4-A, pSⅡRAK-4-B)。质粒转染后24 h,加入0.1 μg/ml脂多糖(LPS)。6 h后,蛋白免疫印迹法及逆转录-聚合酶链反应测定IRAK-4蛋白和mRNA表达水平;酶联免疫吸附法检测0、1、3、6、12 h后KCs的核因子- κB(NF-κB)活性及培养上清液中肿瘤坏死因子α含量。结果RNAi抑制组IRAK-4表达水平,以及LPS刺激后NF-κB活性、肿瘤坏死因子α峰值均明显低于正常对照组和RNAi对照组,t值分别为22.50, 4.18及958.49,P<0.01;尤其是pSⅡRAK-4-A组,抑制效果明显优于pSⅡRAK-4-B,t值分别为12.60, 3.36及256.39,P<0.01。结论以IRAK-4为靶点的shRNA能有效的阻断内毒素诱导的KCs激活效应。
Objective To investigate the feasibility of blocking the activation of Kupffer cells (KCs) induced by endotoxin with interleukin-1 receptor-associated kinase-4 (IRAK-4) short hairpin RNA (shRNA). Methods Two positive plasmids (pSⅡRAK-4-A, pSⅡRAK-4-B) and a pair of negative vector plasmids (pSⅡRAK-4-C) expressing IRAK-4-shRNA were constructed. The KCs were isolated and cultured. The cells were divided into normal control group, RNAi control group (transfected with pSⅡRAK-4 C) and RNAi suppression group (transfected with pSⅡRAK-4-A, pSⅡRAK-4-B). 24 h after transfection, 0.1 μg / ml lipopolysaccharide (LPS) was added. The protein and mRNA expression of IRAK-4 were detected by Western blotting and RT-PCR after 6 h. The expressions of nuclear factor-kappa B NF-κB) activity and tumor necrosis factor-α content in culture supernatant. Results The expression of IRAK-4 and NF-κB in tumor necrosis factor-α (RNAi) group were significantly lower than those in normal control group and RNAi control group (t = 22.50, 4.18 and 958.49, P <0.01) Especially pSⅡRAK-4-A, the inhibitory effect was significantly better than that of pSⅡRAK-4-B, with t values of 12.60, 3.36 and 256.39, respectively, P <0.01. Conclusion shRNA targeting IRAK-4 can effectively block endotoxin-induced KCs activation.