弓形虫排泄分泌抗原对B16F10黑素瘤小鼠CD4^＋CD25^＋Foxp3^＋T细胞和NK细胞的影响

目的探讨弓形虫排泄分泌抗原（ESA）对B16F10黑素瘤小鼠CD4＋CD25＋Foxp3＋T细胞和NK细胞数量和功能的影响,观察ESA对肿瘤生长的作用。方法将传代培养的B16F10黑素瘤细胞接种于C57BL/6小鼠右腋窝皮下,建立荷瘤动物模型。采用ESA腹腔注射进行干预,将实验小鼠分为4组：PBS组、B16F10组、PBS＋ESA组、B16F10＋ESA组,分别于ESA干预后2、4、6d取脾,流式细胞术检测各组小鼠脾脏CD4＋CD25＋Foxp3＋T细胞和NK细胞占脾细胞的比例;WST-8法检测各组小鼠CD4＋CD25＋T细胞对CD4＋CD25-T细胞增殖的抑制功能;LDH法检测NK细胞杀伤B16F10功能;观测荷瘤鼠肿瘤体积动态变化。结果 ESA干预4、6d后,荷瘤鼠脾脏CD4＋CD25＋Foxp3＋T细胞占脾细胞的比例分别为（1.65±0.18）%和（1.56±0.17）%,与荷瘤对照组分别为（2.47±0.10）%和（2.82±0.12）%]比较差异均有统计学意义（P均〈0.05）;ESA干预可使CD4＋CD25＋T细胞对CD4＋CD25-T细胞的抑制功能下降。抑制作用以干预后4、6d较明显,抑制率分别为50.03%和50.00%,低于荷瘤对照组的75.03%和78.14%（P均〈0.05）;荷瘤鼠脾脏NK细胞占脾细胞的比例（3.58±0.07）%]在ESA干预后6d显著高于荷瘤对照组的（2.61±0.13）%（P〈0.05）。同时NK细胞杀伤B16F10细胞功能也增强,不同效靶细胞比（5∶1、10∶1、20∶1）下分别为26.51%、35.25%、60.19%,明显高于荷瘤对照组的16.81%、24.63%、45.62%（P均〈0.05）;ESA干预后,瘤体出现延缓生长,平均出瘤时间较对照组延迟6d,实验终点荷瘤第35天ESA干预组瘤体体积（6208.34±443.64）mm3]明显小于荷瘤对照组（9027.46±1362.01）mm3]（P〈0.05）。结论弓形虫ESA可通过下调荷瘤鼠CD4＋CD25＋Foxp3＋T细胞比例并抑制其功能和上调NK细胞比例并增强其杀伤功能,发挥抗肿瘤作用。

Effects of excreted/secreted antigens of Toxoplasma gondii on CD4+ CD25+ Foxp3+ T cells and NK cells of melanoma-bearing mice

Objective To explore the effects of excreted/secreted antigens （ESA） of Toxoplasma gondii on CD4＋CD25＋ Foxp3＋ T cells and NK cells of melanoma-bearing mice, as well as the tumor growth. Methods B16F10 （denoted B16） tumor cells were cultured in complete medium and maintained by serial passage in vitro. The 2×105 B16 tumor cells were injected into the right flank of the mouse to establish the tumor-bearing mice model. Mice were randomly divided into four groups, namely PBS,B16F10,PBS/ESA and B16F10/ESA groups after ESA injections. On days 2, 4 and 6 post-ESA injection, the spleens were removed. The percentage of CD4＋CD25＋ Foxp3＋ T cells and NK cells in splenocytes were determined by flow cytometry; the suppression functions of CD4＋CD25＋ Tregs and the NK cell activity were detected by WST-8 and LDH methods, respectively. The tumor growth of each group was measured. Results On Days 4 and 6 post-ESA injection, the percentages of CD4＋CD25＋ Foxp3＋ T cells in splenocytes of the B16F10/ESA-injected mice decreased being （1.65±0.18）% and （1.56±0.17）%, respectively, and compared with those in the B16-injected mice （2.47±0.10）% and （2.82±0.12）%], there were significant differences （both P values 0.05）. The inhibition of CD4＋CD25＋ Tregs of the B16F10/ESA-injected mice decreased markedly on Day 4 （50.03%） and Day 6 （50%） compared with those in the control （75.03% and 78.14%） post-ESA injection, there were significant difference （both P values 0.05）. The percentages of NK cells in splenocytes on Day 6 post-ESA injection （3.58±0.07）%] was significantly higher than that of control （2.61±0.13）%]. The activities of NK cells from B16F10/ESA-injected mice against B16 cells at different effect-to-target cell ratios （5∶]1, 10∶]1, 20∶] 1）, increased significantly being 26.51%, 35.25%, 60.19%, respectively, while compared with those in the control （16.81%, 24.63% and 45.62%）, there were significant differences （all P values 〈0.05）. In addition, the volume of the B16 tumors （6 208.34± 443.64）]mm3 was significantly smaller than that of control （9 027.46±1 362.01）] mm3（P〈0.05） when measured at Day 35 post-tumor innoculation. Conclusions T. gondii ESA can downregulate CD4＋CD25＋ Tregs while upregulating NK cells of B16 tumor-bearing mice quantitatively and functionally, therefore plays a role in suppression of tumor growth.