microRNAs在两性胸腺差异性退化中的作用及其基因调控网络研究

Clinically, females have stronger immunity and longer lifespan than males, which may be associated with the slower thymus involution rate in females; the destruction of the epithelial-thymic microenvironment is a major cause of thymus involution; at the molecular level, the thymus involution is the result of gene expression; while, microRNAs (miRNAs) are powerful gene posttranscriptional regulators. Thus, we speculate that miRNAs in thymic epithelial cells (TEC) may exhibit different expression patterns during the process of thymus involution between the sexes, which may contribute to the gender-biased thymus involution. Therefore, by using gene chip and deep sequencing methods, this project attempts to select out differentially expressed miRNAs in females versus males TEC in the process of thymus involution, construct its special expression plasmid or“miRNA sponge”expression plasmid and transfect into in vitro cultured TEC for functional verification, then inject into the thymus to raise or silence the related miRNAs expression within TEC to regulate its target gene expression, thereby inhibiting the thymus involution or promoting thymic regeneration. This project will deeply reveal the sex differences and miRNAs gene regulatory network of thymus involution at the molecular level, and develop new ideas and strategies that using the posttranscriptional regulation of miRNAs on TEC gene expression for the efficient control of thymus involution, as well as establish new ways of inhibiting thymus involution or promoting thymic regeneration.

临床上，雌性比雄性免疫力更强，女人比男人长寿，这可能与雌性胸腺退化更慢相关；上皮性胸腺微环境的破坏是胸腺退化的主要原因；在分子水平上，胸腺退化是基因表达的结果；而microRNAs（miRNAs）是功能强大的基因转录后调节因子。因此，在两性胸腺退化过程中胸腺上皮细胞（TEC）可能存在差异表达的miRNAs，它们调控着胸腺退化及其性别差异。为此，本项目拟应用深度测序等方法筛选出两性小鼠胸腺退化过程中TEC差异表达的miRNAs，构建其过表达质粒或“miRNA海绵”表达质粒，经转染体外培养的TEC进行功能验证后注射入胸腺内，上调或沉默TEC内相关miRNAs以调节其基因表达，从而抑制胸腺退化或促进胸腺再生。本项目将在分子水平深入揭示胸腺退化的性别差异及miRNAs基因调控网络，开辟利用miRNAs在转录后调节TEC基因表达以调控胸腺退化的新思路和新策略，建立抑制胸腺退化或促进胸腺再生的新方法。

本项目的提出是基于这样一个事实，即中枢免疫器官胸腺的退化雌性比雄性更慢。由于在分子水平上胸腺退化是基因选择性表达的结果，而microRNAs是重要的基因转录后调节因子，因此推测在雌、雄胸腺退化过程中可能存在差异表达的microRNAs，它们参与调控胸腺退化及其性别差异。为此，本项目应用高通量深度测序等方法，对雌、雄小鼠胸腺退化过程中胸腺组织和胸腺上皮细胞的microRNAs表达谱进行了深入分析，筛选出了许多与年龄及性别相关的差异表达的microRNAs；同时，还研究了雄激素5α-二氢睾酮、雌激素17β-雌二醇和类雌激素玉米赤霉烯酮对小鼠胸腺上皮细胞增殖凋亡及microRNAs表达的影响，筛选出了许多受雄激素和雌激素调节的microRNAs；并重点研究了miR-16-5p、miR-152-3p、miR-181-5p、miR-183-5p、miR-195a-5p、miR-199b-5p、miR-205-5p、miR-326等microRNAs对小鼠胸腺上皮细胞增殖凋亡的影响。此外，本项目还对雌、雄小鼠胸腺退化过程中差异表达的lncRNAs和mRNAs，以及受雄激素和雌激素调节的胸腺上皮细胞lncRNAs和mRNAs进行了研究。综上所述，本项目在分子水平上深入研究了小鼠胸腺退化的年龄及性别差异，为探索以microRNAs为靶点建立抑制胸腺退化和促进胸腺再生的方法奠定了基础。