miR-449a调控胸腺髓质上皮细胞发育分化的研究

Medullary thymic epithelial cells (mTEC) express and present a diversity of periphery tissue restricted antigens (TRAs), leading to the deletion of self-reactive T cells to establish central immunological tolerance. Accumulating evidence indicated that members of the TNFR family, such as RANK, CD40, and lymphotoxin beta receptor, and the activation of the downstream alternative NF-kappa B signaling pathway are required for the development and differentiation of mTECs and the establishment of central tolerance. However, how alternative NF-kappa B signaling regulates mTEC development is largely unknown. We screened and verified the induction of miR-449a expression by RANK stimulation and activation of alternative NF-kappa B signaling. In addition, our results demonstrated that miR-449a can induce K5＋K8＋ thymic epithelial progenitor cells (TEPCs) to differentiate into Aire-expressing K5＋K8－mature mTECs. This project will generate miR-449a deficient mice and aims to reveal an important function of miR-449a in mTEC development with a new insight into mTEC differentiation and central immunological tolerance, providing a theoretical basis for the development of potential immunointerference strategies based on thymus regeneration and breakdown or re-establishment of central tolerance for the diseases such as autoimmune diseases, organ rejection and tumor.

胸腺髓质上皮细胞（mTECs）表达并递呈外周组织限制性的抗原（TRAs），从而清除自身反应性T细胞，建立中枢免疫耐受。肿瘤坏死因子受体(TNFR)超家族如RANK、CD40、lymphotoxin beta 受体等激活的非经典NF-kappa B信号调控mTECs的发育分化和中枢免疫耐受，但对于非经典NF-kappa B信号下游如何调控胸腺上皮细胞前体细胞（TEPCs）向mTEC的发育分化则不清楚。我们筛选并验证RANK信号及其激活的非经典NF-kappa B信号能诱导miR-449a的表达；在K5/K8双阳性的TEPCs中过表达miR-449a能诱导其分化为表达Aire的K5单阳性的成熟mTECs，提示miR-449a可能调控mTECs发育分化。本项目将通过构建miR-449a敲除小鼠，研究miR-449a在调控mTECs发育分化中的分子机制，揭示mTECs发育分化的程序性事件。本项目将深入理解建立中枢免疫耐受的机制，为以胸腺再生、重建或打破免疫耐受为基础设计针对自身免疫病、器官移植和肿瘤等的免疫干预策略提供理论基础。

胸腺髓质上皮细胞（mTECs）表达并递呈外周组织限制性的抗原（TRAs），在T细胞发育过程中为清除自身反应性T细胞提供分子信号，建立中枢免疫耐受。肿瘤坏死因子受体(TNFR)超家族激活的非经典NF-B信号调控mTECs的发育分化和中枢免疫耐受，但对于非经典NF-B信号下游如何调控胸腺上皮细胞前体细胞（TEPCs）向mTEC的发育分化则不清楚。我们通过microRNA array筛选发现RANK信号及其下游激活的非经典NF-B信号能诱导miR-449a的表达；在TEPCs中过表达miR-449a能诱导其分化为成熟的mTECs，提示miR-449a可能调控mTECs发育分化。本项目通过构建miR-449a敲除小鼠进一步研究miR-449a在调控mTECs分化发育中的分子机制，揭示mTECs发育分化的程序性事件。通过本项目的研究我们发现：1）miR-449a在小鼠胚胎发育的第15.5天开始表达上升并维持较高的表达水平，且miR-449a的表达与Aire的表达水平存在正相关性。2）miR-449a能诱导胸腺上皮前体细胞向成熟的胸腺髓质上皮细胞分化。3）在小鼠体内单独敲除miR-449a不能影响胸腺的发育，但miR-34a在基因敲除小鼠体内表达上升，说明miR-34a可能对miR-449a敲除具有代偿作用。4）在小鼠胸腺原位表达miR-34/449海绵体能抑制成熟的胸腺髓质上皮细胞分化，说明miR-34/449超家族分子对胸腺上皮细胞分化具有重要的调控作用。本项目揭示了miR-34/449家族分子是调控胸腺髓质上皮细胞分化的关键基因。