端粒蛋白Rap1表达和转位与衰老炎症进程关联性研究

Cellular senescence is well-known to be the essential pathology of aging and age-related diseases. Inflammaging is resulted from the switching of cells from growing phenotype to secreting phenotype, which is called the senescence-associated secretory phenotype (SASP)， in response to DNA damage. It has been reported that Rap1 might link telomere length to NF-kB signaling. At long telomere, Rap1 exits within nucleus by binding to telomeres. As a consequence of telomere shortening, Rap1 molecules translocate from nucleus to cytoplasm, concentrate in the cytoplasm and therefore enhance NF-kB signaling through binding and activation of IKKs. Our preliminary data shows that compared to wild type, Rap1-/- Mφ exhibits low responsiveness of cytokine production while high responsiveness of cytoskeleton reorganization stimulated by LPS. Therefore, we formulate a hypothesis that telomeric protein Rap1 expression/translocation accelerates the inflammaging progression. We plan to conduct a series of comparative studies using macrophages of genetically overexpressed or depleted Rap1 as cell models, such as comparative studies on Rap1 expression and location between macrophages from young and elderly mouse/human individuals, cellular behaviors of Rap1-/- Mφ and Mφ from young mice, and cellular behaviors between mouse/human Mφ with ectopic expression of Rap1 and respective Mφ with aging phenotype. The differences of T cell behaviors will also be analyzed in the program due to the modulation of inflammation on T cells. The goal of this study is to provide new targets for treatment and prevention of infammaging.

细胞老化是衰老和衰老相关疾病的病理基础，DNA损伤积累等因素导致细胞老化，演变出"老化相关分泌表型"，衰老炎症是这一演变的结果。有报道指出，端粒缩短时端粒蛋白Rap1转位到细胞浆，增强NF-kB信息传递。我们初步实验表明，与野生型相比，Rap1-/-巨噬细胞（Mφ）对LPS的刺激，细胞因子分泌呈低反应性，而细胞骨架重构呈高反应性。为此，我们提出：由于端粒缩短，Rap1表达和转位加速衰老炎症进程的假说。项目拟采用基因敲除/沉默和转导策略，以小鼠和人的自然状态Mφ和遗传操作的Mφ为研究模型，进行一系列的比较性研究，例如，比较年青和年老鼠/人的Mφ中Rap1的表达水平和细胞内定位；Rap1-/- Mφ与年青小鼠Mφ细胞行为比较；Rap1转导人/鼠Mφ与对应的衰老表型Mφ细胞行为的比较。由于炎症对T细胞的调制作用，T细胞行为差异也属于本项目研究内容。项目的预期结果将为干预衰老炎症提供新的靶蛋白。

项目背景：.细胞老化是衰老和衰老相关疾病的病理基础，DNA损伤积累等因素导致细胞老化，演变出“老化相关分泌表型”，衰老炎症是这一演变的结果。随着年龄的增大，细胞复制的次数越多，染色体端粒的损伤也越明显，进而引发线粒体功能障碍。有报道指出，端粒缩短时端粒蛋白Rap1 转位到细胞浆，增强NF-kB 信息传递。.主要研究内容：.包括体外研究和体内研究。在体外实验中，以免疫细胞（淋巴细胞和巨噬细胞）为研究对象，对比研究了细胞的活化、增殖、分化、凋亡等。包括：衰老和年青的野生型小鼠免疫细胞，Rap1 敲除和野生型的小鼠免疫细胞，老年人和年青人免疫细胞。在体内实验中，向老年鼠和年青鼠以及野生型和Rap1敲除鼠的腹腔中注射高剂量的大肠杆菌建立急性腹膜炎小鼠模型，对比小鼠的存活率以及体内的炎症水平。.重要结果：.1、与年青的野生型相比，年青的Rap1-/-小鼠的巨噬细胞呈现长期存活，对LPS刺激呈现低反应性，低表达协调刺激分子，较高的细胞骨架重构能力，高水平的迁移和吞噬，对钙离子过载引发的凋亡呈现抵抗性；年青的Rap1-/-小鼠的T淋巴细胞对刺激剂引起的活化、增殖和促炎细胞因子分泌呈现低反应性；年青的Rap1-/-小鼠的胸腺细胞的生理性自发凋亡率和地塞米松诱导凋亡率较低；.2、与年青的野生型小鼠相比，衰老的野生型小鼠的T细胞对刺激剂引起的活化、增殖和细胞因子分泌呈现低反应性；.3、与衰老的野生型小鼠相比，衰老的Rap1-/-小鼠巨噬细胞呈现长期存活，低表达协同刺激分子；.4、与年青人相比，老年人的T细胞呈现高活化水平和低增殖能力，分泌较高的促炎细胞因子，表达高水平的凋亡相关分子；.5、与野生型相比，Rap1沉默的人巨噬细胞对LPS刺激呈现反应性；.6、与衰老的野生型小鼠相比，衰老的Rap1-/-小鼠具有较高的胸腺指数；.7、在急性炎症小鼠模型实验中，发现Rap1-/-小鼠的存活率较野生型高，且Rap1-/-小鼠分泌的促炎细胞因子水平较低；发现年青小鼠的存活率较衰老小鼠高，虽然年青小鼠分泌的促炎细胞因子水平较高。.关键数据及其科学意义：.本项目发现Rap1参与了调控免疫功能及炎症水平，为进一步证实“端粒缩短、Rap1转位、NF-kB激活、衰老炎症四者互为因果；Rap1是衰老炎症的加速器”这一工作假说奠定基础，Rap1有望成为继mTOR之后的另一个“成功衰老”的调控靶点。