【作者】 杨牧；

【导师】 周红；

【作者基本信息】 电子科技大学 ， 生物医学工程， 2012， 博士

【摘要】 TGF-β1属于TGF-β超家族，研究表明它在哺乳动物免疫系统中发挥了重要的调节作用，尤其在外周免疫系统中能诱导激活CD4阳性幼T细胞表达Foxp3，从而分化形成具有维持免疫耐受与抑制自免疫反应功能的调节性T（Treg）细胞亚群。相对于哺乳动物中的深入研究，TGF-β1在包括硬骨鱼的低等脊椎动物免疫系统中的功能地位并不清楚。本论文以草鱼外周血白细胞和头肾白细胞为模型，发现TGF-β1对这两群细胞的活性，T/B细胞标志分子（CD4、CD8α/β和Igμ）和效应分子（TNF-α和IFN-γ）的表达分别产生了刺激和抑制两种调节作用。这种相反的调节功能可能与不同模型中的免疫细胞亚群的种类和状态相关。进一步的研究证明，TGF-β1I型受体activin receptor-like kinase5（ALK5）参与了上述双向调节过程。尤其重要的是ALK5mRNA表达与蛋白水平被TGF-β1明显的下调，进而降低了ALK5阳性细胞在各细胞模型中的比例；这一发现被接下来的TGF-β1免疫中和实验所证实，提示一种基于受体层面的TGF-β1信号通路负调控机制的存在。有趣的是，TGF-β1的双向调节功能和降低受体表达作用均是通过短时的‘触发’方式来实现，而不需要长时间的刺激。这一现象可能与下调ALK5表达的机制来共同防止TGF-β1过度调控。基于哺乳动物免疫系统中Foxp3作为TGF-β1免疫抑制功能重要载体的事实，我们在分离克隆草鱼转录因子Foxp3基因的基础上，发现TGF-β1在硬骨鱼白细胞中不仅明显上调Foxp3基因和蛋白表达水平，而且诱导头肾白细胞分化形成Foxp3阳性细胞亚群。研究证实这类细胞与哺乳动物Treg细胞具有相似的免疫抑制功能，具体表现为对激活白细胞活性的抑制以及下调该细胞中效应分子（TNF-α和IFN-γ）基因的表达。对这种现象的相关机制进一步研究证明草鱼Foxp3阳性细胞亚群是通过分泌TGF-β1与IL-10，以非细胞接触形式来发挥免疫抑制的作用。该结果与哺乳动物诱导性Treg细胞的作用特征相符合。综上所述，本论文首先揭示了TGF-β1在鱼类免疫中的双向调节作用，以及其受体层面的自调控机制，其次阐明了硬骨鱼TGF-β1诱导产生Foxp3阳性细胞亚群的免疫抑制功能及其作用机制。研究结果从分子和细胞层面首次明确了硬骨鱼中“TGF-β1-Foxp3-类Treg细胞”调节机制，为鱼类免疫学增添了新内容。更多还原

【Abstract】 TGF-β1belongs to the TGF-β1superfamily, and its pivotal regulatory roles inmammalian immune system have already been extensively characterized. Generally,TGF-β1promotes the expression of Foxp3in activated na ve CD4+cells, resulting inthe generation of the Foxp3+Treg subgroup, which acts as a potent immune suppressivemediator to maintain the immune tolerance and prevent autoimmunity.However, the functional role of TGF-β1in the immune system of teleost is poorlyunderstood. In this study, we examined the immunoregulatory effects of TGF-β1ingrass carp peripheral blood leukocytes (PBL) and head kidney leukocytes (HKL). It isinteresting that TGF-β1consistently stimulated the cell viability and the mRNA levelsof pro-inflammatory cytokines (tnfα and ifnγ) and T/B cell markers [cd4-like (cd4l),cd8α, cd8β and igμ] in PBL, which contrasted with its inhibitory tone in HKL. It seemsthat the bidirectional function of TGF-β1in grass carp may correlate with the oppositestatus of the subsets in PBL and HKL.Further studies showed that grass carp TGF-β1type I receptor, activin receptor-likekinase5(ALK5), was indispensable for the immunoregulatory effects of TGF-β1inPBL and HKL. Notably, TGF-β1persistently attenuated ALK5expression, whereasimmunoneutralization of endogenous grass carp TGF-β1could increase ALK5mRNAand protein levels. It is consistent with the observation that TGF-β1decreased thenumber of ALK5+leukocytes in PBL and HKL, revealing a negative regulation ofTGF-β1signaling at the receptor level. Moreover, transient treatment with TGF-β1for24h was sufficient to induce similar cellular responses compared with the continuoustreatment. This indicated a possible mechanism by which TGF-β1triggered thedown-regulation of ALK5mRNA and protein, leading to the desensitization of grasscarp leukocytes toward TGF-β1.Considering the potential effect of TGF-β1on Foxp3expression and the crucialrole of Foxp3expression in Treg activity in mammals, grass carp foxp3(gcfoxp3) genewas cloned and identified in this study. As expected, TGF-β1was effective in up-regulating gcFoxp3mRNA and protein levels in HKL. Meanwhile, TGF-β1inducedthe generation of grass carp Foxp3+cells. Similar to mammalian Treg, these cellsdisplayed immune suppressive activities in co-culture system in which Foxp3+cellsinduced by TGF-β1down-regulated the cell viability and the expression of effectormolecules (tnfα and ifnγ) of the responder cells stimulated by PHA. A possibleexplanation for the suppressive functions of these Foxp3+cells could be due to thesecretion of cytokines (TGF-β1and IL-10) via a non-cell contact mechanism.Accordingly, our data revealed a dual role of TGF-β1in teleost immunity in whichit can serve as a positive or negative control device and provided additional mechanisticinsights as to how TGF-β1controls its signaling in vertebrate leukocytes. Furthermore,we demonstrated the regulatory mechanism of ‘TGF-β1-Foxp3-Treg-like cells’ axis inteleost.更多还原