CTLA-4 siRNA对慢性乙型肝炎患者外周血Th1/Th2细胞因子的影响

Effect of silencing CTLA4 with siRNA on the Th1/Th2 cytokines in peripheral blood of chronic hepatitis B patient

目的:观察化学合成的小干扰RNA(small interfering RNA,siRNA)对慢性乙型肝炎(chronic hepatitis B,CHB)患者外周血淋巴细胞细胞毒T淋巴细胞相关抗原4(cytotoxic T-lympho-cyte antigen 4,CTLA-4)表达的抑制作用及干扰后对细胞因子IFN-γ、IL-2和IL-4分泌的影响,探讨CTLA-4对慢性乙型肝炎的T细胞免疫调节作用。方法:检测CHB患者外周血淋巴细胞CTLA-4,观察其与HBV-DNA的相关性(P<0.05);根据人淋巴细胞CTLA-4的基因序列,设计合成CTLA-4 siRNA及阴性对照siRNA(siR-NA-co),电穿孔法转染CHB患者外周血淋巴细胞,采用实时荧光定量PCR(real-time Q-PCR)方法检测淋巴细胞CTLA-4 mRNA的表达,采用Western Blotting检测淋巴细胞CTLA-4蛋白表达,采用酶联免疫吸附试验(enzyme-linkedim-muno sorbent assay,ELISA)检测IFN-γ、IL-2和IL-4分泌。结果:CHB患者外周血淋巴细胞CTLA-4表达量与血清HBV-DNA载量有关;CTLA-4 siRNA转染CHB患者外周血淋巴细胞后,CTLA-4 mRNA及CTLA-4蛋白表达均受到抑制,IFN-γ、IL-2分泌增加,与阴性对照相比差异具有统计学意义(P<0.01)。而IL-4分泌没有变化,与阴性对照相比差异没有统计学意义(P>0.05)。结论:CHB患者外周血淋巴细胞CTLA-4表达一定程度抑制免疫反应,利于HBV-DNA的复制;利用siRNA在mRNA水平抑制CHB患者外周血淋巴细胞CTLA-4的表达,能够诱导Th1型细胞因子IL-2、IFN-γ分泌增加,对Th2型细胞因子IL-4无影响。说明抑制CTLA4有助于慢性乙型肝炎患者T细胞免疫的增强。

AIM： To explore the inhibition of expression for cytotoxic T-lymphocyte antigen 4（CTLA-4） in peripheral blood lymphocytes in patients with CHB by chemically synthesized small interfering RNA（siRNA） and the secretion of IFN-γ,IL-2,IL-4,then to analyze the T cellular immune activation by the interference of CTLA-4 gene. METHODS： CTLA-4 of peripheral blood lymphocytes in patients with CHB was detected to observe the correlation with the HBV-DNA;according to human CTAL-4 gene sequence,CTAL-4 siRNA and the negative control（siRNA-co） were designed,synthesized,and then they were transfected into peripheral blood lymphocytes separated from CHB patients by electroporation.The expression of CTLA-4 mRNA was detected by real-time Q-PCR,the CTAL-4 protein was measured by Western Blot and the secretion of IFN-γ,IL-2 and IL-4 were assayed by enzyme-linked immunosorbent assay（ELISA）.RESULTS：CTLA-4 of peripheral blood lymphocytes in patients with CHB was correlated to HBV-DNA（P〈0.05）;after CTLA-4 were transfected into peripheral blood lymphocytes separated from CHB patients,the expression of CTLA-4 mRNA,CTAL-4 protein were suppressed and the production of IFN-γ,IL-2 were enhanced,compared with the negative control group（P〈0.01）,while IL-4 concentrations did not show any significant differences（P〈0.05）.CONCLUSION： CTLA-4 of peripheral blood lymphocytes in patients with CHB might contribute to the persistent infection of HBV.Inhibiting the expression of CTLA-4 mRNA in peripheral blood lymphocytes in patients with CHB could up-regulate the secretion of Th1-type cytokines IFN-γ,IL-2 except Th2-type cytokines IL-4.It suggested that inhibiting CTAL-4 is helpful to activating T cellular immunity of patients with CHB.