摘要
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 contrast HBV transgenic mice with normal mice about the frequency and function of CD4＋ CD25＋ regulatory T cells and its significance. Methods Flow cytometry was used to analyze the proportion of peripheral blood CD4＋ CD25＋ regulatory T cells in the CD4＋ T cells from 18 HBV transgenic mice and 18 normal mice. CD4＋CD25＋ regulatory T cells and CD4＋ CD25- T cells were isolated from HBV transgenic mice splenocytes using a magnetic bead CD4＋ CD25＋ regulatory T cells isolation kit,and then cultured alone or mixed in vitro in the HBsAg （5 mg/L） stimulus group and the non-HBsAg stimulus group. Flow cytometry was used to analyze the frequency of CD4＋ CD25＋ regulatory T cells secreting TGF-β. Lymphocyte proliferation was measured to evaluate the inhibition of CD4＋ CD25＋ regulatory T cells. ELISA was used to detect the level of IFN-γ and IL-6. The expression of hepar Forkhead/winged helix transcription factor （Foxp3） protein was examined by immunohistochemistry. Results There was no significant difference in the proportion of CD4＋ CD25＋ regulatory T cells in the CD4＋ T cells from HBV transgenic mice and normal mice （P0.05）,and there was still no significant difference in the expression level of Foxp3 protein between HBV transgenic mice and normal mice （P0.05）. In the HBsAg stimulus group,the proliferation activity of CD4＋ CD25- T cells from HBV transgenic mice was significantly lower than that in normal mice （P0.05）,and the levels of IFN-γ and IL-6 secreted by CD4＋ CD25- T cells in HBV transgenic mice was significantly lower than that in normal mice （P0.01）. In the non-HBsAg stimulus group,there was no significant difference in the frequency of CD4＋CD25＋ regulatory T cells secreting TGF-β,in the proliferation activity of CD4＋ CD25- T cells and in the level of IFN-γ and IL-6 secreted by CD4＋ CD25- T cells between HBV transgenic mice and normal mice （P0.05）. In all the 2 groups,the proliferation activity of CD4＋ CD25- T cells alone from HBV transgenic mice or normal mice was significantly higher than that mixed culturing （P0.01）,and the level of IFN-γ and IL-6 secreted by CD4＋ CD25- T cells alone from HBV transgenic mice or normal mice was significantly higher than that in mixed culturing （P0.01）. Conclusion There is no significant difference in the frequency and the function of CD4＋ CD25＋ regulatory T cells between HBV transgenic mice and normal mice,but there exists immune tolerance to HBV. CD4＋ CD25＋ regulatory T cells can non-specifically inhibit homebody activated CD4＋ CD25- T cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第14期1499-1503,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(30700709)~~
