Notch信号通路对乙型肝炎患者CD4^+T淋巴细胞分泌白细胞介素22的影响

Influence of Notch signaling pathway on interleukin-22 secreted by CD4^+T cells in patients with hepatitis B

目的观察抑制Notch信号通路对乙型肝炎患者CD4^+T淋巴细胞分泌白细胞介素(IL)22的影响,初步阐释Notch-IL-22信号通路在乙型肝炎发病中的作用。方法收集2013年7月-2014年12月在唐都医院门诊就诊和住院的乙型肝炎初治患者45例,其中急性乙型肝炎(AHB)13例和慢性乙型肝炎(CHB)32例,另收集20例健康志愿者作为对照组(NC组),分离CD4^+T淋巴细胞,应用实时定量PCR法检测Notch1和Notch2 mRNA的表达水平。应用抗CD3抗体或HBV核心区多肽库刺激CD4^+T淋巴细胞,同时加入Notch信号通路抑制剂DAPT,应用实时定量PCR法检测培养细胞中IL-22 mRNA的水平,应用ELISA法检测培养上清中IL-22分泌蛋白的水平。采用Kruskal-Wallis H检验对不同组间数据进行统计分析,Dunn's多重检验对组间数据进行两两比较。结果 Notch1 mRNA在CHB患者CD4^+T淋巴细胞中的表达水平约为NC组的2倍(Z=7.708,P=0.018),Notch2 mRNA的表达水平在AHB和CHB患者中均较NC组升高超过10倍,差异具有统计学意义(Z值分别为9.643、12.900,P值均<0.000 1)。抑制Notch信号通路对培养的CD4^+T淋巴细胞中IL-22 mRNA的表达水平无明显影响(P值均>0.05),但可显著降低NC组(Z=5.068,P=0.015)、AHB组(Z=5.203,P=0.016)和CHB组(Z=2.892,P=0.047)中非特异性IL-22的分泌水平。结论抑制Notch信号通路可阻断病毒非特异性CD4^+T淋巴细胞分泌IL-22,提示Notch-IL-22信号通路在HBV感染中可能发挥促进非特异性炎症应答的作用。

Objective To investigate the influence of Notch signaling pathway inhibition on interleukin- 22（ IL- 22） secreted by CD4^＋T cells in patients with hepatitis B,and to elaborate on the role of the Notch- IL- 22 signaling pathway in the pathogenesis of hepatitis B.Methods A total of 45 previously untreated patients with hepatitis B who visited and were hospitalized in Tangdu Hospital from July 2013 to December 2014 were enrolled,and among these patients,13 had acute hepatitis B（ AHB） and 32 had chronic hepatitis B（ CHB）. Another20 healthy volunteers were enrolled as normal control（ NC） group. CD4^＋T cells were isolated,and quantitative real- time PCR was used to measure the mRNA expression of Notch1 and Notch2. CD4^＋T cells were stimulated by anti- CD3 antibody or HBV core peptide library and the Notch signaling pathway inhibitor DAPT was added. Quantitative real- time PCR was used to measure the mRNA expression of IL-22 in cells,and ELISA was used to measure the level of IL- 22 secretory protein in supernatant. The Kruskal- Wallis H test was used for statistical analysis of data between groups,and the Dunn＇s multiple test was used for data comparison between any two groups. Results The mRNA expression of Notch1 in CD4^＋T cells in CHB patients was about 2 times that in NCs（ Z = 7. 708,P = 0. 018）,and the mRNA expression of Notch2 in AHB and CHB patients was more than 10 times that in NCs（ Z = 9. 643 and 12. 90,both P〈0. 000 1）. Inhibition of the Notch signaling pathway did not influence the mRNA expression of IL- 22 in cultured CD4^＋T cells,but significantly reduced the secretion of non- specific IL- 22 in NCs（ Z = 5. 068,P = 0. 015）,AHB patients（ Z = 5. 203,P = 0. 016）,and CHB patients（ Z = 2. 892,P = 0. 047）. Conclusion Inhibition of the Notch signaling pathway can block IL- 22 secretion by non- specific CD4^＋T cells,suggesting that the Notch- IL- 22 signaling pathway may promote non- specific inflammatory response in HBV infection.