论文部分内容阅读
目的观察急、慢性乙型肝炎(AHB、CHB)患者外周血CD_4~+CD_(25)~(high)曲调节性T细胞(Treg)的频率、表型和功能特点。方法采集16例AHB急性发病期(发病后第1周)患者、72例CHB患者和32例健康人的外周血,检测Treg频率,并分析其表面CD_(45)RO、CD_(45)RA、HLA-DR、CD_(95)和细胞内细胞毒T淋巴细胞相关抗原4(CTLA-4)的表达水平。应用实时荧光定量RT-PCR检测CD_4~+ CD_(25)~+、CD_4~+ CD_(25)~-、CD_4~+和CD_4~-等细胞亚群和外周血单个核细胞(PBMC)的FoxP3 mRNA表达量。通过MACS免疫磁珠分选Treg,并应用[~3H]掺入法检测Treg抑制抗-CD_3抗体和HBV抗原刺激的PBMC增殖能力,并观察Treg对HBV抗原或抗-CD_3抗体刺激自体PBMC分泌IFN_γ的影响。结果CD_4~+CD_(25)~(high)Treg高表达CD_(45)RO、HLA-DR、CD_(95)和细胞内CTLA-4,低表达CD_(45)RA,并且较特异的高表达FoxP3 mRNA。乙型肝炎病人外周血Treg频率与健康对照(3．50±0．72)%比较无统计学差异,但CHB组(3．90±1．44)%显著高于AHB组(3．10±0．87)%,P＜0．05。Treg本身对于HBV抗原或抗- CD_3抗体刺激没有明显的增殖反应和IFN_γ分泌,但可抑制自体PBMC增殖和IFN_γ分泌,其中对HBV抗原刺激引起的细胞反应抑制作用较强。结论HBV感染者外周血Treg较特异地表达FoxP3分子,能抑制HBV抗原特异性细胞免疫反应,这对于深入阐明CHB发病机制具有重要意义。
Objective To investigate the frequency, phenotype and function of CD_4 ~ + CD25 (T) T cells in peripheral blood of patients with acute and chronic hepatitis B (AHB, CHB). Methods The peripheral blood of 16 patients with acute phase AHB (the first week after onset), 72 CHB patients and 32 healthy controls were collected and Tregs were detected. The surface CD45R, CD45RA, HLA-DR, CD_ (95) and intracellular cytotoxic T lymphocyte associated antigen 4 (CTLA-4). Real-time fluorescent quantitative RT-PCR was used to detect the expression of FoxP3 in peripheral blood mononuclear cells (PBMCs) of CD_4 ~ + CD25 +, CD45 + CD25 +, CD45 + and CD45- The amount of mRNA expression. The Tregs were sorted by MACS magnetic beads and the proliferation of PBMC stimulated by anti-CD3 and HBV antigens by Treg was detected by [~ 3H] incorporation method. Tregs were also tested for the secretion of IFN-γ by PBMCs stimulated by HBV antigens or anti-CD3 antibody Impact. Results CD_4 ~ + CD_ (25) ~ (high) Tregs expressed CD45R, HLA-DR, CD_95 and CTLA-4 and CD_ (45) RA, FoxP3 mRNA. The frequency of Treg in peripheral blood of patients with hepatitis B was not significantly different from that of healthy controls (3.50 ± 0.72)%, but significantly higher in CHB patients (3.90 ± 1.44)% than in AHB patients (3.10 ± 0.87)%, P <0.05. Treg itself has no obvious proliferative response and IFN-γ secretion to HBV antigens or anti-CD3 antibody stimulation, but it can inhibit autologous PBMC proliferation and IFN-γ secretion, especially against HBV antigen-stimulated cell response. Conclusion Treg in peripheral blood of HBV-infected patients express FoxP3 specifically, which can inhibit HBV antigen-specific cellular immune responses, which is of great significance for further elucidating the pathogenesis of CHB.