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目的观察弓形虫排泄-分泌抗原(ESA)含糖组分对小鼠CD4+CD25+Foxp3+Treg细胞的影响。方法采用刀豆蛋白A(ConA)琼脂糖凝胶亲和层析技术提取ESA中的含糖组分,经硫酸-苯酚法测定提取物糖含量,并采用BCA法进行蛋白定量测定。将20只雌性C57BL/6小鼠随机分为5组,分别对各组小鼠腹腔注射ESA、ConA结合ESA、ConA未结合ESA、完全RPMI-1640和甘露糖各100μl。4d后处死,流式细胞仪检测各组脾脏CD4+CD25+Foxp3+Treg细胞占脾细胞的比例。结果采用ConA琼脂糖凝胶亲和层析法成功提取弓形虫ESA中含糖成分。与对照组相比(RPMI-1640组和甘露糖组),ESA、ConA结合ESA、ConA未结合ESA注射组小鼠的CD4+CD25+Foxp3+Treg细胞占脾细胞的比例均明显下降(P均<0.01)。结论弓形虫ESA中含糖组分和蛋白组分均能引起小鼠CD4+CD25+Foxp3+Treg细胞占脾细胞的比例下降。
Objective To observe the effects of Toxoplasma gondii-secreting antigen (ESA) sugars on CD4 + CD25 + Foxp3 + Treg cells in mice. Methods The sugar content of ESA was extracted by ConA Sepharose affinity chromatography. The content of sugar in extract was determined by sulfuric acid - phenol method and the protein was determined by BCA method. Twenty female C57BL / 6 mice were randomly divided into five groups. ESA, ConA, ESA, ConA unbound ESA, complete RPMI-1640 and mannose 100μl were intraperitoneally injected into each group. After 4 days, the cells were sacrificed and the percentage of splenic CD4 + CD25 + Foxp3 + Treg cells in the spleen was determined by flow cytometry. Results The sugar content of ESA in Toxoplasma gondii was successfully extracted by ConA agarose affinity chromatography. Compared with the control group (RPMI-1640 group and mannose group), the proportion of CD4 + CD25 + Foxp3 + Treg cells in splenocytes of ESA-ConA-conjugated ESA and ConA-unbound ESA-treated mice were significantly decreased <0.01). Conclusion Both the content of sugar and protein in ESA of Toxoplasma gondii can cause the proportion of CD4 + CD25 + Foxp3 + Treg cells in spleen cells decreased.