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目的比较HBV转基因小鼠和正常同种小鼠CD4+ CD25+调节性T细胞数量和功能的差异及其意义。方法用流式细胞术对18只HBV转基因小鼠和18只正常同种小鼠外周血CD4+ CD25+调节性T细胞的频率进行分析;磁珠分选小鼠脾CD4+ CD25- T细胞和CD4+ CD25+ T细胞,分为HBsAg刺激组和非HBsAg刺激组体外单独和混合培养,分别以流式细胞术、淋巴细胞增殖实验和ELISA检测分泌TGF-β的CD4+ CD25+ T细胞频率,评价CD4+ CD25+调节性T细胞对CD4+ CD25- T细胞增殖的抑制作用,以及诱生的细胞因子IFN-γ、IL-6水平;免疫组化检测肝脏叉状头/翅膀状螺旋转录因子(Forkhead/winged helix transcription factor,Foxp3)蛋白的表达水平。结果与正常同种鼠比较,HBV转基因小鼠外周血CD4+ CD25+调节性T细胞数量无明显差异(P>0.05),肝脏淋巴细胞Foxp3表达也无明显差异(P>0.05)。HBsAg刺激组,HBV转基因小鼠CD4+ CD25- T细胞的增殖能力、诱生的细胞因子IFN-γ、IL-6水平显著低于正常同种鼠(P<0.01);非HBsAg刺激组,HBV转基因小鼠分泌TGF-β的CD4+ CD25+ T细胞数量、CD4+ CD25- T细胞的增殖能力、诱生的细胞因子IFN-γ、IL-6水平与正常同种鼠相比则无明显差异(P>0.05);无论在HBsAg刺激组还是非HBsAg刺激组,两组小鼠的CD4+ CD25- T细胞单独培养时CD4+ CD25- T细胞增殖能力、诱生的细胞因子IFN-γ、IL-6的水平均显著高于混合培养(P<0.01)。结论与正常同种鼠比较,HBV转基因小鼠CD4+ CD25+调节性T细胞数量和功能无明显差异,但在T细胞水平对HBV存在特异性免疫耐受。CD4+ CD25+调节性T细胞可非特异地抑制自身活化的CD4+ T细胞。
Objective To compare the difference of CD4 + CD25 + regulatory T cells and their function between HBV transgenic mice and normal mice. Methods The frequency of CD4 + CD25 + regulatory T cells in peripheral blood of 18 HBV transgenic mice and 18 normal allo-mice was analyzed by flow cytometry. The spleen CD4 + CD25- T cells and CD4 + CD25 + T cells were sorted by magnetic beads The cells were divided into HBsAg-stimulated group and non-HBsAg-stimulated group separately and mixed culture in vitro. The frequency of CD4 + CD25 + T cells secreting TGF-β was detected by flow cytometry, lymphocyte proliferation assay and ELISA, respectively. CD4 + CD25 + regulatory T cells On the proliferation of CD4 + CD25- T cells, as well as the induced cytokine IFN-γ, IL-6 levels; Immunohistochemical detection of liver Forkhead / winged helix transcription factor (Foxp3) Protein expression levels. Results Compared with normal mice, the number of CD4 + CD25 + regulatory T cells in peripheral blood of HBV transgenic mice showed no significant difference (P> 0.05), but there was no significant difference in Foxp3 expression in liver lymphocytes (P> 0.05). HBsAg stimulation group, HBV transgenic mice CD4 + CD25- T cells proliferation, induced cytokines IFN-γ, IL-6 levels were significantly lower than normal (P <0.01); non-HBsAg stimulation group, HBV transgene The number of CD4 + CD25 + T cells secreting TGF-β, the proliferation ability of CD4 + CD25- T cells, the levels of IFN-γ and IL-6 induced by TGF-β in mice were not significantly different from those in normal mice ). The proliferation of CD4 + CD25- T cells and the levels of cytokines IFN-γ and IL-6 induced by CD4 + CD25- T cells from both groups were significantly higher than those from HBsAg-stimulated or non-HBsAg-stimulated groups Higher than mixed culture (P <0.01). Conclusion Compared with normal mice, there is no significant difference in the number and function of CD4 + CD25 + regulatory T cells in HBV transgenic mice, but there is specific immune tolerance to HBV at T cell level. CD4 + CD25 + regulatory T cells can non-specifically inhibit self-activating CD4 + T cells.