摘要
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 explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells （KCs） through short hairpin RNA （shRNA） targeting interleukin-1 receptor associated kinase-4 （IRAK-4） gene. Methods Two effective transfection shRNA plasmid （pSIIRAK-4-A, pSⅡRAK-4-B） and one invalidated plasmids （pSⅡRAK-4-C） targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups： the normal control group, the RNAi control group （pSⅡRAK-4-C） and the RNAi effective group （pSⅡRAK-4-A, pSⅡRAK-4-B）. Then KCs were stimulated with 0.1 1μ g/ml lipopolysaccharide （LPS） after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF- κ B in KCs and the TNF α level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h. Results The level of IRAK-4, the activities of NF- κ B and the TNF-α level in the RNAi effective group were evidently lower than those in normal and RNAi control groups （P 〈0.01） at 1 h, 3 h, and 6 h. Especially, the pSⅡRAK-4-A group in which the changes of the above indices were of no difference （P 〉0.05）, had better inhibited effects than that of the pSⅡRAK-4-B group （P 〈0.01）. Conclusion The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第11期819-822,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金(30471969
30500473)重庆市自然科学基金(2005BB5242)
