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目的在体内外水平研究大蒜新素对小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染导致调节性T细胞(regulatory T cells,Treg)异常扩增的抑制作用。方法 72只MCMV感染小鼠随机分为安慰剂组和大蒜新素治疗组;并设72只同期模拟感染小鼠为对照,随机分为模拟感染对照组和大蒜新素对照组。分别于大蒜新素处理后1、7、14、28、60、120 d处死小鼠,获得脾细胞待测。采用Western blotting法检测脾细胞中Foxp3的蛋白表达强度;流式细胞术分析CD4+CD25+Foxp3+Treg细胞在脾细胞中所占比率的变化。采用小鼠胚胎成纤维细胞对大蒜新素的最大耐受浓度处理MCMV感染的胚胎成纤维细胞3 d后,采用Real-time PCR法和Westernblotting法分别检测T细胞中Foxp3 mRNA和蛋白的表达水平。结果整体研究发现,大蒜新素对正常小鼠脾细胞中Foxp3蛋白表达和Treg细胞比率均无显著影响;但是在MCMV感染小鼠,大蒜新素可在感染慢性期显著减少Foxp3蛋白表达强度、降低调节性T细胞比率。体外细胞模型发现,最大耐受浓度的大蒜新素加入共培养体系后可部分拮抗MCMV诱导的Foxp3基因和蛋白表达上调。结论大蒜新素在体内外均可显著纠正MCMV感染导致的Treg异常扩增。大蒜新素通过影响Treg增殖进而增强抗病毒免疫而有利于机体清除CMV病毒,可能是大蒜新素诱导抗CMV免疫的另一重要机制。
Objective To investigate the inhibitory effect of allitridin on the abnormal amplification of regulatory T cells (Tregs) induced by murine cytomegalovirus (MCMV) infection in vitro and in vivo. Methods Totally 72 MCMV infected mice were randomly divided into placebo group and allitridin treatment group. 72 mice simultaneously infected with MCVV were randomly divided into control group and allitridin control group. The mice were sacrificed at 1,7,14,28,60,120 d after allitridin treatment and the spleen cells were obtained. The protein expression of Foxp3 in spleen cells was detected by Western blotting. The percentage of CD4 + CD25 + Foxp3 + Treg cells in splenocytes was analyzed by flow cytometry. The maximum tolerated concentration of allicin in mouse embryonic fibroblasts was used to treat MCMV-infected embryonic fibroblasts for 3 days. Real-time PCR and Western blotting were used to detect the expression of Foxp3 mRNA and protein in T cells respectively. Results The results showed that allitridin had no significant effect on Foxp3 protein expression and Treg cell ratio in normal mouse spleen cells. However, allitridin significantly decreased the expression of Foxp3 protein and decreased the expression of Foxp3 in MCMV infected mice Regulatory T cell ratio. In vitro cell model found that the maximum tolerated concentration of allitridin added co-culture system can partially antagonize MCMV induced Foxp3 gene and protein expression. Conclusion Allitridin can significantly correct the abnormal amplification of Treg induced by MCMV infection both in vitro and in vivo. Garlicin is another important mechanism by which allitridin can induce anti-CMV immunity by affecting the proliferation of Treg and further enhancing the anti-viral immunity.