结节性硬化TSC1/2复合物对胸腺上皮细胞分化的调控作用及分子机制

The thymus, as a primary immune organ, is critical for the generation of T cells. Thymic epithelial cells (TECs) play important role in supporting the development and maturation of T cells and dysfunction of TECs leads to many immune disease. The development of TECs was regulated by multiple signaling pathways, however, the role of tuberous sclerosis TSC1/2 complex has not been reported. Our recently work by using TEC-specific TSC1 knockout mice showed that deletion of TSC1 led to development blockage of thymus and decrease of TECs, but the molecular mechanism is not clear. We will further clarify the role of TSC1/2 complex on the development of thymocytes and establishment of central immune tolerance and the regulation mechanism of TSC1/2 complex on cortical and medullary TECs by using genetic engineering mice, the preparation and three-dimensional technology. We will recover the regulation network of TSC1/2 complex in TECs by using RNA-seq and bioinformatics analysis. We will elucidate the key regulation pathway by which TSC1/2 complex regulates the differentiation of TECs, by using gene overexpression, Crispr/Cas9 and molecular biology technology. This project will help us understand the development mechanism of thymus and provides new insight to manipulate the development of thymus.

中枢免疫器官胸腺是T细胞产生的重要场所。胸腺上皮细胞（TECs）在T细胞发育分化中发挥重要作用，其异常与许多免疫疾病密切相关。TECs受多种信号通路的精细调控，而结节性硬化TSC1/2复合物在胸腺发育中的作用尚无报道。我们近期应用TEC特异敲除TSC1小鼠初步研究结果表明，TSC1分子缺失导致胸腺发育受阻，TECs分化明显缺陷，但分子机制不详。本课题将利用基因工程改造小鼠、TEC 的分离制备、三维培养等手段，进一步阐明TSC1/2 复合物在胸腺发育及T细胞免疫力和免疫耐受建立中的作用，明确对皮质和髓质TECs分化的作用特点。应用组学及生物信息学技术，初步解析TSC1/2 复合物调控TEC分化的主要分子网络。应用过表达、Cas9、细胞分子生物学及生化等方法，明晰TSC1/2 复合物调控TECs分化详细机制。该研究的顺利实施有助于我们对胸腺发育分子机制的理解，为有效调控胸腺发育提供新思路。

中枢免疫器官胸腺是T细胞产生的场所，胸腺上皮细胞（Thymic epithelial cells, TECs）是胸腺中一群重要的基质细胞，TECs在胸腺细胞的发育分化及成熟中发挥着重要的作用。TECs分为皮质上皮细胞（cortical TECs，cTECs）和髓质上皮细胞（medullary TECs，mTECs），分别介导胸腺细胞的阳性选择和阴性选择。TECs的发育分化受到多种信号通路的调控，而结节性硬化TSC1/2复合物在TECs发育中的作用尚无报道。在本研究中我们利用TECs特异敲除Tsc1的小鼠模型（Tsc1 cKO），研究了TSC1/2复合物在TECs发育分化中的作用。我们发现Tsc1通过抑制溶酶体介导的凋亡通路调控mTECs的稳态。TEC特异敲除Tsc1导致mTECs数量显著减少，对cTECs的数量无显著的影响。Tsc1 cKO小鼠中mTECs发育的缺陷导致胸腺细胞发育阻滞和外周T细胞稳态失衡。Tsc1缺失导致mTECs的凋亡显著增加，而对mTECs的增殖能力无显著影响。通过RNA-seq分析，我们发现Tsc1缺失导致mTECs的溶酶体生物合成通路、细胞代谢通路和凋亡通路显著增加。进一步分析发现，Tsc1缺失导致mTECs中活性氧产生增加和溶酶体功能异常，表现为溶酶体中的蛋白酶cathepsin B和cathepsin L泄露到胞质中，泄露到胞质中的蛋白酶进一步将Bid剪切为活化的tBid，从而引起内源性的细胞凋亡。最后，我们证明通过抑制mTORC1的活性能够部分回复mTECs的发育缺陷和过度凋亡。因此，Tsc1通过mTORC1依赖的方式抑制溶酶体介导的细胞凋亡进而调控mTECs的稳态。