摘要目的观察慢性丙型肝炎患者Notch信号通路分子和Toll样受体4(TLR4)的表达变化以及二者的相互作用对CD14+单核细胞功能的影响. 方法收集2017年8月至10月在陕西省人民医院就诊的慢性丙型肝炎初治患者24例和健康志愿者10名,分选CD14+单核细胞,应用Notch信号通路抑制剂DAPT刺激或使用TLR4 siRNA转染CD14+单核细胞,应用实时定量PCR法检测Notch1、Notch2、Hes1和Hes5 mRNA的水平,应用蛋白质印迹法检测TLR4蛋白水平和NF-κB的磷酸化,应用酶联免疫吸附法检测CD14+单核细胞分泌细胞因子的水平.组间比较采用t检验或配对t检验.结果Notch1 mRNA相对表达量 (3.97±2.03对比0.91±0.76,P<0.01)和Notch信号通路下游分子Hes1 mRNA相对表达量 (5.96±2.31对比0.99±0.45,P < 0.01)、Hes5 mRNA相对表达量(4.31±1.05对比0.84±0.20,P < 0.01)在慢性丙型肝炎患者CD14+单核细胞中的表达较健康志愿者亦显著升高.TLR4 mRNA相对表达量(5.14±1.09对比1.27±0.39)和蛋白水平在慢性丙型肝炎患者CD14+单核细胞中的表达较健康志愿者显著升高(P < 0.01).应用DAPT抑制Notch信号通路可降低慢性丙型肝炎患者CD14+单核细胞中TLR4 mRNA相对表达量(2.58±1.36对比4.34±1.88,P < 0.05)和蛋白的表达和NF-κB的磷酸化,其分泌单核细胞趋化蛋白-1([ 94.32±23.59) pg/ml对比(64.07±9.39) pg/ml,P < 0.01]和白细胞介素-8([ 12.54±4.89) pg/ml对比 (7.92±3.01) pg/ml,P < 0.05]的水平亦显著降低.TLR4 siRNA转染可降低慢性丙型肝炎患者CD14+单核细胞中Notch1 mRNA的表达[2.09±1.72对比3.73±1.75,P < 0.05],亦可降低Hes1 mRNA(2.87±0.84对比5.54±0.97,P < 0.01)和Hes5 mRNA(2.89±0.93对比4.51±1.54,P < 0.01)的表达. 结论慢性丙型肝炎患者中Notch信号通路与TLR4存在相互作用,可促进CD14+单核细胞的功能.

abstractsObjective To observe the expressional changes in Notch signaling pathway and toll-like receptor 4 (TLR4) and their interactions on the functions of CD14+ monocytes in chronic hepatitis C patients. Methods A total of 24 treatment-na?ve chronic hepatitis C cases and 10 healthy individuals, who visited Shaanxi Provincial People's Hospital from August to October 2017, were enrolled. Selected CD14+ monocytes were stimulated by the Notch signaling pathway inhibitor DAPT or transfected with TLR4 siRNA, and the levels of Notch1, Notch2, Hes1 and Hes5 mRNA were detected by real-time quantitative PCR. TLR4 protein levels and phosphorylation of NF-κB was detected by Western blot. ELISA was used to detect the level of cytokines secreted from CD14+ monocytes. A t-test or paired t-test was used for comparison between groups. Results The relative expression of Notch1 mRNA (3.97 ± 2.03 vs. 0.91 ± 0.76, P < 0.01) and downstream of Notch signaling pathway (5.96 ± 2.31 vs. 0.99 ± 0.45, P < 0.01), Hes1 mRNA and Hes5 mRNA (4.31 ± 1.05 vs. 0.84 ± 0.20, P < 0.01) in CD14+ monocytes of chronic hepatitis C patients was significantly higher than that of healthy individuals. The relative expression of TLR4 mRNA (5.14 ± 1.09 vs. 1.27 ± 0.39) and protein level in CD14+ monocytes of chronic hepatitis C patients were significantly higher than those of healthy individuals (P < 0.01). An inhibition of Notch signaling pathway with DAPT had reduced the relative expression level of TLR4 mRNA (2.58 ± 1.36 vs. 4.34 ± 1.88, P < 0.05), protein expression and phosphorylation of NF-B in CD14+ monocytes of chronic hepatitis C patients. Furthermore, the secretion level of MCP-1 [(94.32 ± 23.59) pg/ml vs. (64.07 ± 9.39) pg/ml, P < 0.01] and IL-8 [(12.54 ± 4.89) pg/ml vs. (7.92 ± 3.01) pg/ml, P < 0.05] was significantly reduced. TLR4 siRNA transfection reduced the expressions of Notch1 mRNA (2.09 ± 1.72 vs. 3.73 ± 1.75, P < 0.05), Hes1 (2.87 ± 0.84 vs. 5.54 ± 0.97, P < 0.01), and Hes5 (2.89 ± 0.93 vs. 4.51 ± 1.54, P < 0.01) in CD14+ monocytes of chronic hepatitis C patients. Conclusion Interaction of Notch signaling pathway with TLR4 can promote the function of CD14+ monocytes in chronic hepatitis C patients.