天花粉蛋白通过影响Foxp3的泛素化水平增强CD4^(+)Treg的体外诱导的初步研究

Trichosanthin enhances CD4^(+)Treg induction in vitro by affecting the ubiquitination of Foxp3

为研究中药天花粉蛋白(trichosanthin,Tk)能否在体外条件下促进CD4^(+)Treg定向诱导分化及其效率,并初步探讨Tk的负向免疫调节作用与Foxp3分子泛素化水平调控的可能机制,建立经典Treg诱导体系,即分离小鼠脾脏淋巴细胞并加入抗CD3和抗CD28抗体以及IL-2、TGF-β,共同诱导Treg。接着,向培养体系中加入Tk(1.5μg/mL)连续培养3 d,FACS检测发现加入Tk后Treg百分比显著上升(P<0.05);Treg特异性转录因子叉状头转录因子3(forkhead box protein 3,Foxp3)的蛋白表达水平显著上升(P<0.05);Real-Time PCR检测显示其mRNA水平降低显著(P<0.05)。进一步的研究建立过表达Foxp3的HEK293T细胞系,Western blotting检测结果显示,Tk可延长Foxp3蛋白的半衰期(P<0.05),并降低其泛素化水平。以上结果提示,Tk具有负向免疫调节作用,并能提高Treg的百分比及抑制Foxp3蛋白的泛素化,起到稳定Foxp3分子的作用。

A classical Treg induction system was established to investigate how trichosanthin(Tk)regulates the differentiation of CD4^(+)Treg and its efficiency in vitro as well as to explore the negative immunomodulatory effect of Tk and its regulatory mechanism of Foxp3 ubiquitination.Mouse spleen lymphocytes were isolated and Treg was induced by adding anti-CD3 mAb,anti-CD28 mAb,IL-2,and TGF-β.After 3 days of continuous culturing with Tk(1.5μg/mL),FACS analysis showed that the percentage of Treg and the protein level of forkhead box protein 3(Foxp3)increased significantly(both P<0.05).Real-Time PCR indicated that the Foxp3 mRNA level decreased significantly(P<0.05).To explore the underlying mechanism,HEK293T cell line stably overexpressing Foxp3 protein was established.Western blotting showed a prolonged half-life of Foxp3 protein(P<0.05)and decreased ubiquitination level in Tk treated cells.These results suggest that Tk has a negative immunomodulatory effect,possibly by promoting differentiation towards Treg and inhibiting the ubiquitination of Foxp3,thus stabilizing the protein.