慢性乙型肝炎患者CD4^(+)Th细胞中LSD1表达及其作用

Expression of LSD1 in CD4^(+)Th cells and its effect in the patients with chronic hepatitis B

目的:探讨慢性乙型肝炎(乙肝)患者外周血CD4^(+)Th细胞内赖氨酸特异性组蛋白去甲基化酶1(histone lysine specific demethylase 1,LSD1)表达水平与Th1/Th2平衡的关系,初步探讨LSD1介导的组蛋白修饰对慢性乙肝患者CD4^(+)Th细胞分化的作用。方法:选择2017年6月至12月在南通大学附属医院感染科住院的慢性乙肝患者65例,分为丙氨酸转氨酶(ALT)高水平[>4×ULN(正常上限)]组与低水平(≤4×ULN)组,HBV-DNA高载量(≥10^(6)拷贝/mL)组与低载量(<106拷贝/mL)组。以同期在该院体检的30例健康人为对照。采用密度梯度离心法分离外周血单个核细胞,免疫磁珠法分选CD4^(+)Th细胞;提取CD4^(+)Th细胞总蛋白,以Western印迹法检测LSD1表达水平。采用ELISA法检测血清中干扰素γ(IFN-γ)、白细胞介素4(IL-4)含量;用微粒子化学发光法检测血清乙型肝炎表面抗原(HBsAg)含量;用实时定量PCR法检测血清HBV-DNA载量。结果:慢性乙肝组患者外周血CD4^(+)Th细胞中LSD1表达水平高于健康人(0.52±0.21 vs 0.28±0.09,t=-7.49,P<0.001)。慢性乙肝患者中,CD4^(+)Th细胞中LSD1表达量在ALT高水平组(n=38)低于低水平组(n=27,0.39±0.18 vs 0.64±0.16;t=-5.79,P<0.001),在HBV-DNA高载量组(n=32)高于HBV-DNA低载量组(n=33,0.69±0.08 vs 0.35±0.16;t=10.80,P<0.001)。与健康人相比,慢性乙肝组血清IFN-γ含量降低、IL-4含量增高、IFN-γ/IL-4比值减小(P<0.05)。慢性乙肝患者外周血CD4^(+)Th细胞中LSD1表达量与其血清ALT水平(r=-0.590)、IFN-γ水平(r=-0.379)及IFN-γ/IL-4(-0.285)负相关(P<0.01),与HBV-DNA载量正相关(r=0.880,P<0.001),与血清IL-4水平无相关性(r=0.169,P=0.102)。结论:LSD1高表达可能是慢性乙肝患者体内Th1/Th2失衡、Th1反应水平下降的原因之一,并导致机体清除HBV能力减弱或抑制HBV复制能力减弱。

Objective:To explore the relationship between the histone lysine-specific demethylase 1(LSD1)expression in CD4^(+) Th cells and Th1/Th2 cell immune balance of the patients with chronic hepatitis B(CHB),and to explore the effect of histone modifications mediated by LSD1 on the cellular differentiation of CD4^(+) Th cell in patients with CHB.Methods:A total of 65 patients with CHB hospitalized in the Department of Infectious Disease,Affiliated Hospital of Nantong University,from June to December in 2017 were selected and further divided into different groups according to the serum levels of ALT or HBV-DNA load.30 healthy persons who received health physical examination during the same period were as the control group.The peripheral blood mononuclear cells were separated with the density gradient centrifugation,and then CD4^(+) Th cells were sorted using immunomagnetic beads.The total protein of CD4^(+) Th cells was extracted,the expression of LSD1 was detected using Western blotting.The serum levels of interferon-γ(IFN-γ)and interleukin-4(IL-4)were detected using ELISA.The serum HBV-DNA load was detected using real-time qPCR.Results:The expression level of LSD1 in CD4^(+) Th cells in the CHB group was higher than that in the control group(0.52±0.21 vs 0.28±0.09,t=-7.49,P<0.001).In patients with CHB,the expression level of LSD1 in the high ALT level(>4×ULN)group was lower than that in the low ALT level(≤4×ULN)group(0.39±0.18 vs 0.64±0.16,t=-5.79,P<0.001);the expression level of LSD1 in the high HBV-DNA load(≥10^(6) copy/mL)group was higher than that in the low load(<10^(6) copy/mL)group(0.69±0.08 vs 0.35±0.16,t=10.80,P<0.001).Compared with the control group,the serum level of IFN-γand the ratio of IFN-γ/IL-4 was reduced and the serum level of IL-4 was ascending in patients of the CHB group(P<0.05).The expression level of LSD1 in CD4^(+) Th cells in CHB patients was negatively correlated with the serum levels of ALT(r=-0.590),IFN-γ(r=-0.379),and the ratio of IFN-γ/IL-4(r=-0.285,P<0.01),was positively correlated with HBV-DNA load(r=0.880,P<0.001),and was uncorrelated with the serum level of IL-4(r=0.169,P=0.102).Conclusions:In the patients with CHB,the high expression of LSD1 could be one of the reasons for the imbalance of Th1/Th2 reaction and descending of Th1 reaction,which can attenuate the elimination of HBV or the inhibition of HBV replication.