中药天花粉蛋白增强诱导CD4^(+)CD25^(+)Foxp3^(+)调节性T细胞数量和功能的初探

Study on the Effects of Trichosanthin on the Enhanced Induction of the Number and Function of CD4^(+)CD25^(+)Foxp3+Regulatory T Cells

目的探讨中药天花粉蛋白(Trichosanthin,Tk)增强诱导CD4^(+)CD25^(+)Foxp3^(+)调节性T细胞(regulatory T cell,Treg)数量和功能的作用。方法研磨6-8周C57BL/6小鼠脾脏,裂解红细胞后得到单个脾脏淋巴细胞悬液,按5×10^(5)/mL细胞密度均匀接种在圆底96孔板,设空白组、对照组(增殖体系:1μg·mL^(-1) anti-CD3/28 mAb)、三个不同诱导方案组,培养7天后收集细胞运用流式细胞术检测CD4+CD25+Foxp3+Treg细胞比例以此确定最佳诱导方案;将低浓度Tk(0.25、0.5、1μg·mL^(-1))作用于诱导体系,7天后采用流式检测Tk对CD4^(+)CD25^(+)Foxp3^(+)Treg细胞比例的影响;Annexin V-PI染色法检测细胞凋亡情况;Western blotting法测定各组中Treg细胞特异性转录因子Foxp3蛋白表达情况;实时荧光定量PCR法检测转录因子Foxp3和细胞因子TGF-β、IL-10、IL-6、IFN-γ基因水平的表达;酶联免疫吸附法(ELISA)测定各组细胞培养上清中分泌IFN-γ和IL-10的浓度。结果与对照组相比,添加TGF-β(5ng·mL^(-1))诱导小鼠脾脏淋巴细胞生成Treg的数量和比例最高,此诱导方案最佳(P<0.01);与诱导组相比,低浓度中药天花粉蛋白可显著增强诱导CD4+CD25+Foxp3+Treg细胞数量和比例,呈剂量依赖性(P<0.01),且Tk需在诱导体系下发挥作用,单加不能诱导生成Treg细胞,此浓度Tk无细胞毒性;与对照组相比,诱导组和低浓度Tk组Treg特异性转录因子Foxp3蛋白和基因表达水平均显著上调(P<0.01),II型辅助性T细胞(Th2)分泌的炎症抑制因子IL-10、TGF-β基因表达不同程度上调,反之,I型辅助性T细胞(Th1)分泌的促炎因子IFN-γ、IL-6基因表达呈相反趋势下调(P<0.01);与对照组相比,诱导组和低浓度Tk组细胞上清中分泌的IL-10浓度显著增加,IFN-γ浓度明显降低(P<0.01)。结论低浓度中药天花粉蛋白可显著增强诱导CD4^(+)CD25^(+)Foxp3^(+)调节性T细胞,上调Foxp3和Th2型炎症抑制因子IL-10、TGF-β的表达,下调Th1型促炎因子IFN-γ、IL-6的表达,从而发挥免疫抑制功能。

Objective To investigate the effect of Trichosanthin(Tk)on the enhanced induction of the number and function of regulatory T cells(Treg).Methods Spleen lymphocytes were isolated from 6-8 weeks C57BL/6 mice after RBC lysis.And we obtained single spleen lymphocyte suspensions.Cells were sorted by pressing 5×105/mL cell density were seeded evenly in round bottom 96 well plates,the blank,the control,and three different induction regimen groups.No cytokines were added into the blank group,the control group was cultured under the proliferation system by stimulation of 1μg·mL^(-1) anti-CD3/28 mAb.The inducing group was treated with TGF-βor IL-2.The amounts of CD4^(+)CD25^(+)Foxp3^(+)Treg cells were detected by flow cytometry after 7 days in order to determine the best induction method.Then Tk(0.25,0.5,1μg·mL^(-1))was added into the best induction system,and the amount of Treg cells were detected by flow cytometry.Apoptosis was detected by Annexin V-PI staining.The gene and protein expression of Foxp3 which was the specific transcription factor of Treg cells was determined by PCR and Western blotting after Tk treatment.The gene levels of TGF-β,IL-10,IL-6 and IFN-γwere detected by real-time PCR.The concentrations of IFN-γand IL-10 in the supernatant of cell culture were determined by ELISA.Results Compared with the control group,adding TGF-β(5 ng·mL^(-1))induced the highest number and proportion of murine splenic lymphopoietic Treg cells,and this induction regimen was optimal(P<0.01).Compared with the inducing group,it was found that low concentration of trichosanthin significantly promoted the induction of Treg cells in dose-dependent manner only in the inducing system(P<0.01).Moreover,Tk could only function in the inducible system and cannot induce Treg cells alone.This concentration of Tk was not cytotoxic.Compared with control group,the gene and protein expression of specific transcription factor Foxp3 was significantly up-regulated in the induction group and the low concentration Tk group as well as TGF-βand inflammatory factors IL-10 which Th2 cell secreted(P<0.01).The pro-inflammatory factors IL-6 and IFN-γwere down-regulated in the Tk-induced group(P<0.01).Compared with control group,the secretion of IL-10 was significantly increased in the Tk-induced group,while IFN-γwas significantly reduced(P<0.01).Conclusion Low-concentration trichosanthin can significantly enhance the induction of CD4^(+)CD25^(+)Foxp3^(+)Treg cells,up-regulate the expression of Foxp3 and inflammatory suppressor factors TGF-βand IL-10,down-regulate the expression of proinflammatory factors IL-6 and IFN-γ,and thereby play an immunosuppressive role.