含穿膜肽重组转录因子FOXN1-TAT对衰老小鼠胸腺重建的作用及其细胞和分子机制的研究

Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T cell generation. However, TECs undergo degeneration over time, which leads to age-related thymic involution. Enhancing the development of TECs has become the focus of the study on the regeneration of the aged thymus, which is crucial to improve the immune competence of the aged. FOXN1 plays a pivotal role in TECs development. FOXN1 transgenic mice or injecting the FOXN1 plasmid into thymus have been conducted by several research groups. However, the two methods are difficult to control the level and duration of FOXN1 protein expression as well as the attendant risk of insertional mutagenesis. The use of FOXN1 protein may overcome these adversities. The protein transduction domain of TAT is cell-penetrating peptide with high efficiency. The recombinant protein FOXN1 (rFOXN1) fused with TAT cell-penetrating peptide has been expressed successfully in our group, what’s more, rFOXN1 is able to penetrate into cells and translocate to the nucleus efficiently. In this study the optimum scheme of injecting rFOXN1 protein into mice thymus will be determined primarily. The thymic architecture, the quantity and ratio of the total and all subgroups of thymocytes will be examined subsequently in aged mice treated with injecting rFOXN1 intrathymically. Further the quantity and ratio of TEC and its subgroups will be detected thoroughly. At the same time some specific proteins and a series of genes associated with the development of TECs should be investigated intensively. Ultimately the function of the regenerated thymus will be valued. Collectively, this study will answer the question whether the injection of FOXN1 protein into the aged mice intrathymically can mediate the thymic regeneration and reveal the corresponding mechanism, providing the theoretical and experimental basis for improving immune competence of the aged people and preventing geriatric diseases clinically.

胸腺上皮细胞TECs是胸腺微环境主要组分，随年龄增长逐渐退化，直接导致增龄性胸腺退化。促进TECs发育已成为研究衰老胸腺重建的热点，对增强老年人免疫力有重要意义。FOXN1蛋白在TECs发育中起关键作用。有研究制作FOXN1转基因鼠或胸腺内注射FOXN1质粒，但不易控制蛋白表达且有潜在基因突变风险，直接注射蛋白或可克服此问题。TAT的蛋白转导结构域是高效穿膜肽。我们已高效表达重组蛋白FOXN1-TAT且明确了其穿透细胞入核的效率。本课题从探索蛋白进入小鼠胸腺最佳方案入手，重点研究胸腺注射蛋白后，衰老小鼠胸腺形态结构、胸腺细胞及各亚群的改变，并深入探讨衰老小鼠胸腺内TECs及各亚群的量、比例以及与之相关的一系列基因表达情况，再检测重建后胸腺的功能。本研究将明确胸腺注射FOXN1-TAT蛋白能否重建衰老胸腺，揭示相应作用机制，为临床提高老年人免疫力，预防老龄化疾病提供一定的理论和实验依据。

胸腺上皮细胞（Thymic Epithelia Cells, TECs）是胸腺微环境主要组分，随年龄增长逐渐退化，直接导致增龄性胸腺退化。促进TECs发育已成为研究衰老胸腺重建的热点，对增强老年人免疫力有重要意义。FOXN1蛋白在TECs发育中起关键作用。本研究高效表达重组蛋白-TAT（rFOXN1），且明确了其穿透细胞膜并进入细胞核的效率。用rFOXN1体外处理TECs，发现可以有效提高其增殖能力并维持其功能。通过胸腺注射rFOXN1处理衰老小鼠后，小鼠胸腺得以重建，即胸腺体积增大，重量增加，形态结构有较大改善，胸腺细胞及各亚群均明显增加；处理后衰老小鼠的胸腺功能增强，表现为脾脏内执行免疫应答的初始T细胞数目增多，其中近期胸腺迁移显著增加。发生这一转变的机制在于：FOXN1蛋白的增多使衰老小鼠胸腺内TECs及各亚群的比例趋向正常，数量均显著增加，与TECs功能相关的一系列基因的表达得到加强。本研究明确了胸腺注射rFOXN1蛋白可使衰老胸腺重建，并阐述了相应作用机制，为临床提高老年人免疫力，预防老龄化疾病提供一定的理论和实验依据。同时，本研究还发现，在体外细胞水平抑制GSK-3β调控WNT信号通路，可以增加TECs的FOXN1的表达，促进TECs增殖以及增强其功能；体内用GSK-3β小分子抑制IM-12处理衰老小鼠，也能促进TECs增殖，从而有利衰老胸腺的重建以及T细胞再生。这亦为临床缓解衰老个体的胸腺衰退，增强老年人的免疫力提供了新的思路。