靶向沉默Notch1、2和3基因对动脉粥样硬化患者巨噬细胞Notch和核因子-κB信号通路的影响

摘要

目的 探讨靶向沉默Notch 1、2和3基因对动脉粥样硬化患者巨噬细胞内Notch信号通路关键基因Delta-like 4(DLL4)、Jagged 1和核因子-κB (NF-κB)信号通路关键基因IκBα、p52表达的影响,从基因层面研究防治动脉粥样硬化的方法.方法 采集冠状动脉粥样硬化患者静脉血后体外分离单核细胞,并诱导转化为巨噬细胞.将巨噬细胞分为5组:空白对照组(未转染siRNA)、阴性对照组(转染NC-siRNA)、Notch1-siRNA组(转染Notch1-siRNA)、Notch2-siRNA组(转染Notch2-siRNA)和Notch3-siRNA组(转染Notch3-siRNA).分别采用RT-PCR和Western blot法检测各组DLL4、Jagged 1、IκBα和p52的mRNA和蛋白相对表达水平,采用电泳迁移率变动分析(EMSA)检测各组NF-κB活性,采用免疫荧光法检测各组巨噬细胞内p52的分布.结果 (1)RT-PCR和Westernblot检测显示,Notch1-siRNA组、Notch2-siRNA组和Notch3-siRNA组DLL4、Jagged 1和p52的mRNA和蛋白相对表达水平均低于空白对照组和阴性对照组,而IκBα的mRNA和蛋白相对表达水平均高于空白对照组和阴性对照组(P＜0.05或0.01);Notch1-siRNA组DLL4、Jagged 1和p52的mRNA和蛋白相对表达水平均低于Notch2-siRNA组和Notch3-siRNA组,IκBα的mRNA和蛋白相对表达水平均高于Notch2-siRNA组和Notch3-siRNA组(P＜ 0.05或0.01);空白对照组和阴性对照组DLL4、Jagged 1、IκBα和p52的mRNA和蛋白相对表达水平差异均无统计学意义(P均＞0.05).(2)EMSA检测提示,Notch1-siRNA组(613±57)、Notch2-siRNA组(1 169±85)和Notch3-siRNA组(1 454±90)的NF-κB活性均低于空白对照组(2 642±115)和阴性对照组(2 407±100)(P均＜0.01);Notch1-siRNA组的NF-κB活性低于Notch2-siRNA组和Notch3-siRNA组,Notch2-siRNA组的NF-κB活性低于Notch3-siRNA组(P均＜0.01);空白对照组和阴性对照组的NF-κB活性差异无统计学意义(P＞0.05).(3)巨噬细胞核内和胞浆内NF-κB/p52蛋白的总荧光强度Notch1-siRNA组、Notch2-siRNA组和Notch3-siRNA组均低于空白对照组和阴性对照组(P均＜0.01),且Notch1-siRNA组、Notch2-siRNA组和Notch3-siRNA组细胞核内的荧光强度均低于核外(P＜0.01或0.05).空白对照组和阴性对照组细胞核内外的NF-κB/p52蛋白荧光强度差异无统计学意义(P＞0.05).结论 冠状动脉粥样硬化患者巨噬细胞中Notch信号通路与NF-κB信号通路之间存在正向调节,Notch1基因较Notch2和Notch3基因对NF-κB信号通路的调节作用更显著.%Objective To investigate the effects of Notch1,2,3 genes silencing by siRNA on Notch signaling pathway (Delta-like 4 (DLL4),Jagged 1 (JAG1)) and nuclear factor-κB (NF-κB) signaling pathway (IκBα,P52) of macrophages derived from patients with coronary artery disease (CAD),thus to explore the potential genetic treatment perspectives for CAD.Methods Peripheral blood mononuclear cells of CAD patients were isolated by density gradient centrifugation and transformed by phorbol-12-myristate-13-acetate (PMA) to macrophages.Macrophages were then transfected with Notch1-small interference RNA (siRNA,Notch1-siRNA group),Notch2-siRNA (Notch2-siRNA group),Notch3-siRNA (Notch3-siRNA group),negative control siRNA (NC group) and none siRNA (control group) respectively.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were applied to assess the mRNA and protein expression levels of DLL4,JAG1,IκBα and p52,respectively.Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity.Subcellular distributions of NF-κB/p52 were detected through immunofluorescence.Results (1) The mRNA and protein expressions of DLL4,JAG1 and p52 in Notch1-siRNA group,Notch2-siRNA group and Notch3-siRNA group were significantly downregulated,while the mRNA and protein expression of IκBα was significantly upregulated compared with NC group and control group (P ＜ 0.05 or 0.01).The mRNA and protein expressions of DLL4,JAG1 and p52 in Notch1-siRNA group were significantly downregulated,while the mRNA and protein expression of IκBα was significantly upregulated compared with Notch2-siRNA group and Notch3-siRNA group(P ＜0.05 or 0.01).The mRNA and protein expressions of DLL4,JAG1,IκBα and p52 were similar between NC group and control group (all P ＞ 0.05).(2) The binding activity of NF-κB DNA was significantly lower in Notch1-siRNA group (613 ± 57),Notch2-siRNA group (1 169 ± 85) and Notch3-siRNA group (1 454 ± 90) compared with control group (2 643 ± 115) and NC group (2 407 ± 100) (all P ＜0.01),which was also significantly lower in Notch1-siRNA group compared to Notch2-siRNA group and Notch3-siRNA group (P ＜ 0.01);was significantly lower in Notch2-siRNA group compared with Notch3-siRNA group (P ＜ 0.01) and was similar between control group and NC group (P ＞ 0.05).(3) The fluorescence intensity of NF-κB/p52 was significantly lower both in the nucleus and cytoplasm in Notch1-siRNA group,Notch2-siRNA group and Notch3-siRNA group compared with NC group and control group (all P ＜0.01),and the decrease was more obviously in the nucleus than in cytoplasm in Notch1-siRNA group,Notch2-siRNA group and Notch3-siRNA group (P ＜ 0.05 or 0.01).The fluorescence intensity of NF-κB/p52 was similar between control group and NC group (P ＞ 0.05).Conclusion There is a positive regulation between Notch and NF-κB pathway in macrophages derived from CAD patients,the regulation power on NF-κB signaling pathway of Notch1 is stronger than that of Notch2 and Notch 3.