大蒜新素抑制小鼠巨细胞病毒感染诱导调节性T细胞扩增的体外研究

目的 在细胞水平研究大蒜新素对小鼠巨细胞病毒(MCMV)感染鼠胚肺成纤维细胞(MEF)诱导调节性T细胞(Treg)异常扩增的抑制作用.方法 建立MEF和T淋巴细胞的共培养体系,采用MEF对大蒜新素的最大耐受浓度(MTC)处理MCMV感染的MEF 3 d后,再用实时定量PCR检测T细胞中叉头蛋白3(Foxp3)mRNA表达水平,流式细胞术检测效应性T细胞哑群杀伤性T细胞Ⅰ型(Tc1)、杀伤性T细胞Ⅱ型(Tc2)、辅助性T细胞Ⅰ型(Th1)和辅助性T细胞Ⅱ型(Th2)的百分比,双抗体夹心酶联免疫吸附法检测共培养体系中自细胞介素10(IL)-10和转化生长因子(TGF)-β的蛋白表达,标准蚀斑法检测共培养体系中的病毒负荷量,并与安慰剂组进行统计学分析比较.结果 MEF对大蒜新素的MTC浓度为9.83μg/ml.MTC浓度的大蒜新素可部分拮抗MCMV诱导的Foxp3基因表达上调(87±5比114±8,P<0.01);分别上调Tc1、Tc2和Th1百分比至(12.42±1.23)%、(4.28±0.56)%、(13.25±0.68)%,与安慰剂组(6.85±0.92)%、(2.34±0.42)%、(9.32±0.86)%]比较差异均有统计学意义(均P<0.01);使IL-10和TGF-β蛋白表达水平分别降至(29.98±3.15)pg/ml和(3.48±0.23)ng/ml,均显著低于安慰剂组水平(38.21±4.02)pg/ml和(5.31±0.59)ng/ml,均P<0.05];同时将体系中的病毒负荷量由(6.79±0.39)降至(5.03±0.08)(均P<0.01).结论 大蒜新素在体外可通过抑制Treg途径来增强抗病毒免疫.

Role of allitridin in the blocking of murine cytomegalovirus induced regulatory T cells amplification in vitro

Objective To investigate the effect of allitridin on murine cytomegalovirus (MCMV) induced regulatory T cells (Treg) amplification in vitro.Methods A co-culture system of T cells and MCMV infected mouse embryo fibroblasts (MEF) was established.A maximum tolerance concentration (MTC) of allitridin was added into the co-culture system.After 3 days, the change of Foxp3 mRNA was measured by real-time PCR.And the percentages of Tc1 ( CD3 + CD8 + IFN-γ+ ), Tc2 (CD3 + CD8 + IL-4 + ), Th1 ( CD3 +CD8 - IFN-γ+ ) and Th2 ( CD3 + CD8 - IL-4 + ) were analyzed by flow cytometry.The production of IL-10 and TGF-β in supernatants was detected with double-antibody sandwich ELISA while the viral load of culture quantified by plaque assay.All results were compared with those of the placebo group.Results The MTC of allitridin was 9.83 μg/ml in MEF.The treatment of 9.83 μg/ml allitridin could partly block the MCMV induction of Foxp3 mRNA expression (87 ± 5 ) vs ( 114 ± 8), P < 0.01 ].The percentages of Tc1, Tc2 and Th1 significantly increased to the levels of (12.42 ± 1.23)%, (4.28 ±0.56)% and (13.25 ±0.68)%respectively.They showed statistic differences with those of placebo controls (6.85 ± 0.92) %, (2.34 ±0.42)% and (9.32 ±0.86)% ; all P <0.01 ].Meanwhile, the levels of IL-10 and TGF-β1 in supernatants also significantly decreased to ( 29.98 ± 3.15 ) pg/ml and ( 3.48 ± 0.23 ) ng/ml by allitridin treatment as compared with placebo controls (38.21±4.02) pg/ml and (5.31 ±0.59) ng/ml, all P <0.05].In addition, the MCMV plaque assays showed that allitridin significantly suppressed the viral loads by one order of magnitude.Conclusion Allitridin can partly retrieve MCMV-induced Treg expansion and Treg-mediatad anti-MCMV immunosuppression so as to enhance the specific cellular immune responses against CMV.