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

发布时间：2018-03-13 20:13

本文选题：Notch　切入点：NF-κB　出处：《中华细胞与干细胞杂志(电子版)》2016年06期 　论文类型：期刊论文

【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,P0.001),且较空白及阴性对照组均出现明显下降(P0.05),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,P0.001),并均明显高于空白、阴性对照组(P0.05),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,P0.001)。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。
[Abstract]:Objective to investigate the role of Notch and NF- 魏 B signaling pathway in the pathogenesis of atherosclerosis. After transfection with Notch1-siRNA-Notch2-siRNA-Notch3-siRNA and NC-siRNA, the expression of P65 I 魏 B 伪 gene and protein was detected by RT-PCR and Western Blot. The activity of NF- 魏 B was detected by EMSA method. The distribution of p65 in macrophages was detected by immunofluorescence assay. T test was used to compare the difference. Results the results of RT-PCR showed that the expression of p65 gene in Notch1-siRNA group decreased gradually (0.709 卤0.007 卤0.557 卤0.031 卤0.014 卤0.114 卤0.014 4) after transfection of siRNA into macrophages, and there was a significant difference between the two groups in the expression of p65 gene in the Notch2-siRNA group (0.709 卤0.007 卤0.031 卤0.114 卤0.014), and there was a significant difference between the two groups in the expression of p65 gene in the ntch2-siRNA group (0.709 卤0.007 卤0.031 卤0.114 卤0.01414, respectively), and there was a significant difference between the two groups. In negative control group, there was a significant decrease in P0.05 and Noch1-siRNA. The expression level of I 魏 B 伪 increased gradually (1.811 卤0.172 卤3.253 卤0.169 卤5.295 卤0.433), and the difference between the two groups was statistically significant. The expression of P65 I 魏 B 伪 protein in Noch1-siRNA group was the same as that in RT-PCR group. The activity of NF- 魏 B in macrophages transfected with Notch-siRNA was significantly lower than that in control group and negative control group. The fluorescence intensity of p65 protein in nucleus and cytoplasm of macrophages in Notch1-siRNA group (21 405.20 卤929.91) and Notch2-siRNA group (25 987.13 卤911.404.67 卤452.16) was significantly lower than that in control group (55,238.33 卤1 059.21) and negative control group (55 844.27 卤1331.10) (P < 0.01). Conclusion there is a positive regulation between Notch and NF- 魏 B classic pathway in macrophages of as patients, and the effect of Notch1 pathway on NF- 魏 B classic pathway is more significant than that of Notch2ntch3.
【作者单位】：扬州大学附属泰州市人民医院胸痛中心;
【基金】：泰州市社会发展基金(81570748)
【分类号】：R54

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