靶向沉默动脉粥样硬化患者巨噬细胞中Notch基因对NF-κB经典通路的影响  

Effects of silencing Notch genes on NF-κB classic pathway in macrophages of patients with atherosclerosis

作　　者：李伟[1] 朱莉 阮中宝 任寅 王斌[1] Li Wei Zhu Li Ruan Zhongbao Ren Hn Wang Bin.(Department of Cardiology, Taizhou People＇s Hospital, Taizhou 225300, Chin)

机构地区：[1]扬州大学附属泰州市人民医院胸痛中心,泰州225300

出　　处：《中华细胞与干细胞杂志（电子版）》2016年第6期327-333,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：泰州市社会发展基金(81570748)

摘　　要：目的探讨Notch和NF-κB信号通路在动脉粥样硬化(AS)发病过程中的作用方式、两者是否存在串话。方法将AS患者外周血单核细胞诱导为巨噬细胞后,分别经Notch1-siRNA、Notch2-siRNA、Notch3-siRNA以及NC-siRNA转染。使用RT-PCR和Western Blot法检测转染后各组P65、IκBα基因以及蛋白表达情况。EMSA法检测转染后各组NF-κB活性。免疫荧光法检测巨噬细胞内P65分布。两组间资料差异比较采用t检验,多组间比较采用单因素方差分析。结果 RT-PCR结果显示,siRNA转染巨噬细胞后,Notch3-siRNA组、Notch2-siRNA组、Notch1-siRNA组中P65基因表达水平渐次降低(分别为0.709±0.007、0.557±0.031、0.114±0.014),组间差异有统计学意义(F=709.224,P<0.001),且较空白及阴性对照组均出现明显下降(P<0.05),Noch1-siRNA组下降最显著;而IκBα表达水平渐次升高(分别为1.811±0.172、3.253±0.169、5.295±0.433),组间差异有统计学意义(F=112.290,P<0.001),并均明显高于空白、阴性对照组(P<0.05),也以Noch1-siRNA组变化最明显。Western-Blot方法检测所得P65、IκBα蛋白表达结果与RT-PCR结果一致。Notch-siRNA转染后的巨噬细胞内NF-κB活性均显著低于空白对照组和阴性对照组,Notch1-siRNA组活性下降最显著。巨噬细胞核内及胞浆内P65蛋白的荧光强度Notch1-siRNA组(21 405.20±929.91)、Notch2-siRNA组(25 987.13±911.40)、Notch3-siRNA组(28 074.67±452.16)较空白对照组(57 238.33±1 059.21)以及阴性对照组(55 844.27±1331.10)均不同程度减弱(P均<0.01),且细胞核内荧光强度较核外更低,以Notch1-siRNA组减弱最明显(组间比较F=55.137,P<0.001)。结论 AS患者巨噬细胞中Notch与NF-κB经典通路之间存在正向调节,且Notch1通路较Notch2、Notch3对NF-κB经典通路的调节作用更显著。Objective To investigate the mechanism and crosstalk between Notch and NF-~B signaling pathway in macrophages of patients with atherosclerosis （AS）. Methods The mononuclear cells from peripheral blood of patients with AS were induced into macrophages, which were then silenced by Notchl-siRNA, Notch2-siRNA, Notch3-siRNA and NC-siRNA. RT-PCR and Western blot were applied to assess the expression levels of P65 and IκBα in the NF-κB signaling pathway. Electrophoretic mobility shift assay （EMSA） was used to observe the NF-κB DNA binding activity. Subcellular distributions of P65 was detected with immunofluorescence, t test was used to compare the difference of data between two groups. One-way analysis of variance was used to compare the difference among groups. Results RT-PCR results showed that, after Notch-siRNA being transfected into macrophages, the expression of P65 declined in the Notch-3 siRNA group （0.709± 0.007）, the Notch-2 siRNA group （0.557±0.031）, and the Notch- 1 siRNA group （0.114± 0.014） （P 〈 0.01）, all of which were lower than the control （1.004±0.105） group and the negative control group （1.043±0.096） （P 〈 0.05）.The expression of IkBα （1.811±0.172, 3.253±0.169, 5.295 ± 0.433） increased （P 〈 0.01）, all of which were greater than the control （1.003 ± 0.087） group and the negative control group （1.027 ±0.052） （P 〈 0.05）, especially in Notchl-siRNA group. The results of Western Blot were in accordance with those of RT-PCR. The NF-κB DNA binding activity were inhibited. The fluorescence intensity of P65 decreased both in the nucleus and cytoplasm compared to the control group and the negative control group （P 〈 0.01）, which declined more obviously in the nucleus. Conclusions A positive relationship may exist between Notch signaling pathway and NF-κB classic pathway in the macrophages of patients with AS. Notchl pathway may play a more important role in the regulation on NF-κB signaling pathway than Notch2 and Notch 3.

关 键 词：NOTCH NF-ΚB 基因表达 动脉粥样硬化 巨噬细胞 

分 类 号：R54[医药卫生—心血管疾病]