【作者】 李敏；

【导师】 汪洌； 王建莉；

【作者基本信息】 浙江大学 ， 免疫学， 2013， 博士

【摘要】 白细胞介素2(IL-2)是由活化T淋巴细胞分泌的具有免疫调控作用的细胞因子,对于维持免疫稳态具有重要作用。IL-2在体外可促进T细胞活化和增殖,是所有T细胞亚群生长因子,故又称为T细胞生长因子；IL-2还可维持自然杀伤性(NK)细胞、细胞毒性T淋巴细胞(CTL)以及B细胞的增殖分化作用；在体内IL-2同时具有抑制T淋巴系统过度活化和促进外周免疫耐受等功能。IL-2的表达在各个水平上都受到精密调控,过高或过低均会引起体内多种免疫细胞功能紊乱或自身免疫性疾病等。IL-2mRNA在静息T细胞中几乎无法检测到,但在受到刺激活化后短时间即可明显上调,有关其mRNA水平的调控主要取决于其转录和降解两个方面,目前IL-2转录水平调控的研究已经非常深入,而对其mRNA转录后稳定性的调控研究成为一个新的研究热点。转录后调控对于维持那些快速诱导的mRNA的稳态来说非常重要,与其他早期免疫应答基因或生长调控基因一样,IL-2mRNA的转录后稳定性调控主要与其mRNA3’非翻译区(3’UTR)的调控元件相关。单核细胞趋化蛋白诱导蛋白-1(MCPIP1)是最近发现确定的一类具有免疫调节作用的CCCH型锌指家族分子。MCPIP1缺陷小鼠具有严重的自身免疫性疾病,大多数在出生后12周内死亡,其巨噬细胞在TLR信号激活后IL-6和IL-12p40等炎性因子分泌明显增多。目前MCPIP1研究主要集中在负向调控巨噬细胞介导的固有免疫应答过程中,且机制主要是通过与IL-6等细胞因子mRNA3’UTR结合,发挥其N端RNA酶活性,引起整条1mRNA的降解,从而下调靶基因表达。我们研究发现MCPIP1在T细胞活化后短时间内可被诱导,并可负向调控T细胞重要细胞因子IL-2的表达水平。我们构建了T细胞特异性MCPIP1转基因小鼠,发现该小鼠外周T细胞数目有一定程度减少,其活化后分泌IL-2水平明显降低；之后在鼠源以及人外周血CD4+T细胞体外过表达或干扰MCPIP1后,发现其可通过影响IL-2mRNA半衰期从而负向调控其表达；进一步机制探讨发现MCPIP1是通过靶向IL-23’UTR(83-166)一段非AU富集区(ARE)的保守区所形成的茎环结构从而引起整条mRNA的降解。综上所述,我们寻找到在转录后水平调控T细胞关键性细胞因子IL-2的RNA结合蛋白-MCPIP1,进一步完善了IL-2的调控网络,同时也对MCPIP1在适应性免疫应答中的作用提供了新的启发。更多还原

【Abstract】 Interleukin2(IL-2), a key T lymphocytes-derived immunoregulatory cytokine, plays a major role in maintaining lymphocyte homeostasis. IL-2stimulates the activation, survival and proliferation of T lymphocytes in vitro, while its main function in vivo is to limit lymphoid expansion and to promote peripheral tolerance. The absence of IL-2results in the development of lethal autoimmunity and abnormal high level of IL-2impairs the functions of many immune cells. The IL-2mRNA achieves its normal level in the stimulated T lymphocytes by regulating the rate of transcriptional activation and mRNA degradation. The posttranscriptional regulation is via controlling the stability of mRNA. IL-2is known to be regulated by RNA binding proteins, such as tristetraprolin (TTP), via an AU-rich element (ARE) in the3’-untranslated region (3’UTR) to influence the stability of mRNA.The CCCH zinc finger protein MCPIP1(encoded by ZC3H12A gene) is previously known as a negative regulator in the macrophage activation. MCPIP1knockout mice developed a syndrome of severe anemia or severe autoimmune response and most of the mice died within12weeks. Macrophages from MCPIP1knockout mice showed highly increased production of IL-6and IL-12p40in response to TLR ligands. The underlying mechanism is mainly considered that MCPIP1is an essential RNase and down-regulates specific mRNAs of cytokines via a conserved region in the non-ARE of the3’UTR. Here, we reported that MCPIP1was induced in the activation of T lymphocytes and negatively regulated IL-2gene expression in both mouse and human primary T lymphocytes through destabilizing its mRNA. We generated a transgenic mouse model of MCPIP1overexpression, in which MCPIP1was expressed in a T cell-restricted fashion, and we found that IL-2expression of transgenic mouse T cells was much lower than that of wild type mouse T cells. A set of luciferase reporter assay demonstrated that a non-ARE conserved element in IL-23’UTR, which formed a stem-loop structure, responded to MCPIP1activity. RNA immunoprecipitation and Biotin pulldown experiments further suggested that MCPIP1could modestly bind to IL-2mRNA. Taken together, these data demonstrate that MCPIP1down-regulates IL-2via an ARE-independent pathway.更多还原