Notch信号在胸腺上皮细胞退化中的作用机制及强力霉素对其调控机制的研究

Age-associated and stress-induced involution of the thymus is accompanied by a decline in the number of thymic epithelial cells (TEC), the decrease of thymic output and a severely restricted peripheral repertoire of T-cell specificities. These events affect the immune system function seriously, but the mechanisms are unclear. Beverly and his colleagues found that T cells, overexpression of the Notch ligand Jagged1 in the transgenic mouse, induced TEC apoptosis. We also found that Jagged1 can induce the TEC apoptosis when a mouse TEC cell-line MTEC1 cells co-cultured with Jagged1 recombination protein. Doxycycline (Dox), a tetracycline antibiotic, has been shown to have neuroprotective efficiency in reduction of a variety of ischemia-reperfusion injuries as well as ischemic brain damage via inhibiting the MMPs, PKCδ and caspase. Recently, we found that Dox not only increase ERK to promote the proliferation of MTEC1 cells, but upregulate the secretion of IL-6 and GM-CSF via NF-κB and ERK. It also increases the expression of BCL2, decreases the expression of p53, inhibits the activation of Notch, and protects MTEC1 resistance to the cell apoptosis induced by mitomycin C or Jagged1 protein. Therefore, we propose a hypothesis: "The Notch signaling initiates the TEC apoptosis in thymic involution of age-associated or stress-induced; Dox downregulates the activation of Notch signal in TEC and protects them from apoptosis, then attenuates the degradation and atrophy of the thymus with aging or stress". In this study, we used the hematopoietic stem cell transplantation and Notch ligands to activated the Notch signal in TEC to investigate the mechanisms of Notch signal pathway in degradation of the thymus, and to declare the mechanism of Dox of protecting TEC. It will be beneficial to T-cell reconstitution and develop a set of new therapeutic drugs for clinical.

TEC的增龄性和应激性退化导致T细胞特异性库容和输出量不可逆减少，严重影响机体免疫功能，但机制不详。Beverly发现过表达Jagged1的T细胞诱导TEC凋亡，我们也发现了相似情况。Dox具保护神经细胞抵抗凋亡作用。我们发现Dox不仅上调ERK和NF-κB促进胸腺上皮细胞系（MTEC1）增殖、分泌IL-6和GM-CSF；而且可上调BCL2，下调p53的表达，抑制Notch的活化和保护MTEC1抵抗丝裂霉素C和Jagged1诱导的凋亡。故我们提出假说："Notch信号介导了TEC的增龄性及应激性退化；Dox能下调TEC的Notch信号活化，保护胸腺上皮细胞，减缓胸腺增龄性或应激性退化和萎缩"。本研究采用Notch配体或造血干细胞移植活化TEC的Notch信号，研究Notch在胸腺退化中的作用机制，并阐明Dox对该信号的调控机制，为临床进行T细胞重建和治疗胸腺退化药物的研发奠定实验基础。

胸腺是机体T细胞发育的重要场所，但胸腺很早就开始退化和萎缩。目前国内外研究认为其原因主要有三个方面，①生长激素的下降，性激素的升高；②造血干细胞的减少、自我更新减弱和归巢障碍；③最为重要的是由胸腺上皮细胞（TEC）构成的T细胞发育分化的微环境的改变，其中关键转录因子Foxn1表达明显下调，但确切机制尚不清楚。文献报道胸腺细胞过表达Jagged1的转基因小鼠中，出现胸腺萎缩。我们前期研究发现活化Notch信号可以诱导TEC凋亡。故我们推测Notch信号可能与TEC凋亡相关。以往的研究表明活化的T细胞、树突状细胞（DC）均可表达Jagged1，因此我们假设在感染等状态下，活化的免疫细胞迁移至胸腺内，与TEC表面Notch分子结合诱导TEC的死亡。但由于存在血-胸腺屏障，回迁的细胞数量很少，因此这一过程非常缓慢，但在生命过程中却可能起着重要作用。本课题从①不同年龄段小鼠TEC表达Jagged1等Notch受体水平与胸腺萎缩的相关性分析；②体外活化Notch信号后TEC的生物学行为；③Notch途径活化诱导TEC的凋亡机制；④强力霉素对TEC的保护机制等四个方面进行了研究。研究初步结果表明：①髓质区TEC表达Notch3分子更为明显，随着年龄的增长表达水平下降；②活化Notch信号在体外可诱导TEC的凋亡；③活化T细胞（以Th1为代表）可通过分泌IFN-γ诱导TEC的凋亡；④强力霉素可以通过上调Trx2-NF-κB-Bcl-2信号途径保护TEC。本研究初步结果表明胸腺増龄性萎缩可能与活化的免疫细胞回迁导致TEC的凋亡有关，同时强力霉素对保护TEC具有一定的作用。这一成果将为深入揭示胸腺退化的分子机制，研发强力霉素等新的药理作用和机制或新药研发，延缓胸腺功能退化，降低老年肿瘤和自身免疫病的发病奠定基础。.总之，本项目研究基本达到了原定目标，初步阐明的了胸腺退化，特别是胸腺上皮细胞退化导致的胸腺萎缩的机制。但是仍然许多有不足之处：如，①未能在体内全面完成体外的实验结果；②强力霉素的作用精准靶点尚未能确定。