硒蛋白SelK对小胶质细胞和单核细胞吞噬和迁移能力的影响及与阿尔茨海默病关系研究

Selenoprotein SelK is one of the selenoproteins expressed highly in the brain of mice.The activated macrophages predominantly express the uncleaved SelK and the resting macrophages and other cells predominantly exprss cleaved SelK. Complete SelK can increase the release of Ca2+ from endoplasmic reticulum in macrophages and increase the ability of phagocytosis and migration of macrophages. Previous studies show that the bone marrow-derived monocytes can be recruited into brain and are more efficient in the cleaning up of Aβ protein. Microglia is the 'macrophage' in the brain and monocyte is the precursor cell of macrophage, it is not repored whether SelK can also exist in two states in the activated and resting microglias and bone marrow-derived monocytes and whether the cleaving state of SelK can influence the intracellular free calcium level, phagocytosis and migration ability of the cells, and whether the expression and the cleaving state of SelK related to the onset of Alzheimer's disease. To resolve the problems, the SelK in the microglias and monocytes of mouse was knocked down or over expressed to study the relationship of SelK expression and the level of intracellular free calcium and the ability of phagocytosis and migration. The expression level and the cleaving state of SelK in the brain cells of mouse were also detected. The SelK knock-out mice and SelK recombinant adenoviral expressing vector were constrcuted to study the relationship of SelK and the onset or prevention of AD form the perspective of Immunology.

硒蛋白SelK是小鼠脑中含量较高硒蛋白之一，SelK在活化巨噬细胞中保持完整而在静息态巨噬细胞和其它细胞中被剪切，完整SelK能促进内质网中钙离子释放，提高吞噬、迁移能力，已有研究显示血液来源单核细胞能被动员进入脑部，清除Aβ蛋白更为有效。小胶质细胞是脑中的"巨噬细胞"，单核细胞是巨噬细胞前体,它们在阿尔茨海默病（AD）发生中具重要作用。SelK是否在活化态和静息态小胶质细胞和单核细胞中也保持两种状态，其剪切状态和表达量是否影响到细胞中游离钙离子水平、吞噬、迁移能力，以及是否与AD的发生有关，尚未见相关报道。为此本项目拟通过对小鼠小胶质细胞和单核细胞进行SelK基因敲减和过表达，阐明SelK与上述细胞胞浆中游离钙离子水平、吞噬、迁移能力间关系；另外对小鼠脑部不同细胞SelK表达水平、剪切状态进行测定；构建SelK敲除小鼠和SelK重组腺病毒表达载体，从免疫角度研究SelK与AD发生间关系。

硒蛋白SelK是脑中表达量较高硒蛋白之一，SelK的表达对巨噬细胞吞噬和迁移能力具有重要影响。小胶质细胞是脑中"巨噬细胞"，它们在阿尔茨海默病（AD）发生中具重要作用。微量元素硒是否通过SelK在AD发生过程中发挥作用，SelK的敲减/过表达是否影响小胶质细胞的吞噬和迁移能力，其信号通路是什么、以及SelK是否与AD发生有关，是本项目拟解决的关键科学问题。本项目分别通过细胞实验及SelK敲除小鼠对上述问题进行了研究。研究表明，通过小鼠硒蛋白mSelK过表达或敲减腺病毒表达载体侵染小胶质细胞进行mSelK基因敲减/过表达，显著降低/增加小胶质细胞吞噬和迁移能力。信号通路研究表明，mSelK敲减/过表达显著降低/增加内质网钙离子释放通道蛋白IP3R表达，细胞浆中游离钙离子水平显著降低/升高。对小鼠小胶质细胞进行亚硒酸钠处理，显著增加mSelK表达及细胞内游离钙离子水平。同时构建mSelK敲除小鼠，进行Aβ1-42及AlCl3脑部注射，水迷宫实验表明，AD发生几率显著升高。由此得出以下结论：硒主要通过SelK影响到细胞内游离钙离子水平对小胶质细胞发生影响，SelK表达升高/降低通过调控IP3R升高/降低，引起细胞中游离钙离子水平升高/降低，进一步引起小胶质细胞吞噬和迁移能力升高/降低，进而影响小胶质细胞对Aβ多肽的清除能力发挥作用。小鼠中mSelK表达降低将使小鼠更易患AD，补硒有助于防止AD发生。上述研究揭示了小胶质细胞及硒蛋白SelK在AD发生过程中的重要作用及人体补硒的重要意义，有助于AD的防治。本项目还进一步发现SelK敲减/过表达降低/增加胰岛细胞中IP3R3表达，引起细胞内游离钙离子、胰岛素分泌量降低/升高，证明补硒对防止糖尿病的发生具有重要意义。发表学术论文10篇。1篇SCI索引论文正在大修，可取得后续成果2-3项。