小剂量环磷酰胺对正常小鼠CD4^+CD25^+Treg细胞的调节作用

Regulatory effect of low dose cyclophosphamide on CD4^+CD25^+ Treg cells in mice

目的比较多次与单次应用小剂量环磷酰胺(CTX)对小鼠CD4+CD25+调节性T细胞(Treg)数量及功能的影响,明确小剂量CTX可否多次用于下调Treg细胞。方法 C57BL/6小鼠32只,随机分为正常对照、单次、两次及三次CTX注射组共4组,各组每10天腹腔注射20 mg/kg CTX或等体积PBS,末次给药后3 d,流式细胞技术测小鼠脾细胞中Treg细胞占CD4+T细胞百分比;同时3-2,5-二苯基四氮唑溴盐(MTT)法比较各组脾细胞中T、B细胞增殖功能,中性红吞噬试验比较各组巨噬细胞吞噬功能。结果各实验组小鼠脾细胞中Treg细胞占CD4+T细胞百分比显著低于对照组(P<0.05),而各实验组间差异无统计学意义(P>0.05)。各实验组T、B细胞增殖指数及巨噬细胞吞噬功能明显高于对照组(P<0.05),但各实验组间差异无统计学意义(P>0.05)。应用CTX后Treg细胞数量与T、B细胞增殖功能及巨噬细胞吞噬功能呈线性负相关。结论多次与单次小剂量应用CTX对小鼠Treg细胞数量的下调作用无差异,下调Treg细胞后对免疫细胞的调节功能也无差异,小剂量CTX可多次用于下调正常小鼠Treg细胞。

In order to compare the number and function alteration of CD4＋CD25＋ regulatory T cell treated with low dose cyclophosphamide in mouse,and determine whether low dose cyclophosphamide can be used to down regulate Treg cell repeatedly,thirty-two C57BL /6 mice were randomly divided into four groups： control,once,twice and thrice treated groups.Mice in groups were injected intraperitoneally with 20 mg/kg Cyclophosphamide（CTX） or equal volume PBS every ten days.Three days after the last injection,the ratio of Treg cells to CD4＋ T cells in spleen was detected by flow cytometry;the proliferation of T and B lymphocytes were detected by 3-2,5-diphenyl-tetrazolium bromide（MTT） test;and the phagocytosis of macrophages was detected by neutral red test.Compared to control group,the percentage of Treg cell in CD4＋T cells of treated groups decreased sharply（P 0.05）,but there was no difference between the treated groups（P 0.05）.The proliferation of T and B lymphocytes and the phagocytosis of macrophages were all enhanced,as compared with control group（P 0.05）,but there was no difference between the treated groups（P 0.05）.We also found that the number of Treg cells negatively correlated with the proliferation of T/B lymphocytes and the phagocytosis of macrophages after administrating with CTX.All these results indicate that there is no difference in decreasing Treg cell and regulating the function of immunocells subsequently between the single and multi-administration with low dose cyclophosphamide.Thus low dose cyclophosphamide could be used to down regulate Treg cell repeatedly.