小鼠硒蛋白mSelK及硒对小胶质细胞的作用及与阿尔兹海默病的关系研究

发布时间：2018-10-21 12:01

【摘要】：阿尔茨海默症(Alzheimer's disease, AD)又称老年痴呆症,是一种危害人类身体健康和生命质量,影响家庭和社会的重大疾病,随着我国进入老龄化社会,相关研究更具有现实意义。小胶质细胞是脑内的巨噬细胞,具有免疫活性,发挥着内源性免疫防御作用和保护神经元等功能。在阿尔茨海默病的发生早期,小胶质细胞吞噬和迁移能力的降低,不能及时清除沉积的Aβ蛋白,被认为是阿尔茨海默病发生的重要原因之一,因此提高小胶质细胞的吞噬和迁移能力具有重要意义。硒在人类和动物生命个体中以硒蛋白的形式表达,硒蛋白SelK是脑中含量较高的硒蛋白之一,已有研究发现硒蛋白SelK能够通过促进内质网上控制钙释放主要受体之一的IP3 Receptor (IP3R)的过表达促进内质网中储存钙离子的释放,使胞浆中游离钙离子含量的提高。硒蛋白mSelK和硒是否会影响小胶质细胞的迁移和吞噬能力是急需解决的重要问题。首先构建了小鼠硒蛋白mSelK ( mouse selenoprotein K )过表达的腺病毒载体及基因敲减腺病毒载体及其阴性对照,空白腺病毒载体对照等5种腺病毒载体。采用MTT法检测5种腺病毒载体对静息态和与LPS激活的小鼠小胶质细胞BV2活力的影响。结果显示5种腺病毒载体对静息态和活化态小胶质细胞的存活率均无显著影响,可进行下一步实验研究。采用Transwell小室法和划痕愈合实验法考察了 5种腺病毒载体对静息态和LPS激活小鼠小胶质细胞BV2迁移能力的影响。结果显示,无论Transwell小室实验还是划痕愈合实验均表明硒蛋白mSelK的过表达/基因敲减能够显著提高/降低静息态和激活状态小胶质细胞的迁移能力,且迁移能力大概增高或降低到180%/40%。采用吞噬鸡血红细胞实验法检测了 5种腺病毒载体对小鼠小胶质细胞BV2吞噬能力的影响。结果表明硒蛋白mSelK的过表达/基因敲减能使静息态和LPS激活小胶质细胞的吞噬鸡血红细胞能力显著增加/降低,且吞噬和率吞噬指数分别增加或降低至为180%,5%和200%,3%。综上所述,硒蛋白mSelK对小胶质细胞有着重要的作用,硒蛋白mSelK表达的增加显著提高小胶质细胞的吞噬和迁移能力,而硒蛋白mSelK表达的减少显著降低小胶质细胞的吞噬和迁移能力。为探讨硒蛋白mSelK表达对小胶质细胞吞噬和迁移能力的影响是否是引起小胶质细胞的活化而导致的,通过Griess法(检测NO释放)探讨了 5种腺病毒载体对小鼠小胶质细胞BV-2活化状态的影响。实验结果表明,在引起小胶质细胞吞噬和迁移能力增加及降低的硒蛋白mSelK过表达/基因敲减腺病毒载体侵染滴度下,只有LPS激活小胶质细胞的活化程度随着硒蛋白的过表达/敲减而增加或减少,说明只有活化态小胶质细胞吞噬和迁移能力的增加/减少是由于小胶质细胞活化状态的改变而导致的。而静息态小胶质细胞吞噬和迁移能力的改变并不是由于mSelK表达的变化影响到了小胶质细胞的活化状态。其次采用流式细胞仪方法研究了硒蛋白mSelK的过表达/基因敲减对BV2胞浆中游离Ca2+水平的影响。研究发现,无论对于静息态还是活化态的小胶质细胞,硒蛋白mSelK的过表达均引起胞浆中游离Ca2+的水平的升高至300%;而硒蛋白mSelK的敲减引起胞浆中游离Ca2+水平的降低至60%。另外采用qPCR和Western Blot考察了硒蛋白mSelK对内质网上控制钙离子通道开放关键蛋白酶IP3R (IP3 receptor)表达的影响。研究发现无论对于静息态的还是活化态的小胶质细胞,mSelK的过表达引起细胞内IP3R基因表达的增多;而mSelK的敲减引起细胞内IP3R基因表达的减少。最后研究了亚硒酸钠对小胶质细胞的影响及与硒蛋白mSelK表达之间的关系。MTT实验结果显示,对于静息态小胶质细胞BV-2来说,低于或等于3μM的亚硒酸钠对其生长无抑制,超过此浓度就会抑制细胞生长或引起细胞死亡。采用Griess法检测亚硒酸钠作用于BV-2细胞对细胞活化状态的影响。实验结果表明,亚硒酸钠对静息态小胶质细胞的活化状态没有显著的影响。采用实时定量PCR和Western Blot证实硒蛋白mSelK的表达随着亚硒酸钠的增多而增多,且有剂量依赖的效应。综合以上结果可以得出如下结论:微量元素硒的变化可以增加小鼠小胶质细胞中硒蛋白mSelK表达的变化。小鼠小胶质细胞中硒蛋白mSelK的表达量的升高或降低通过增加或减少内质网上钙离子通道蛋白IP3R的表达,从而引起细胞浆中游离钙离子水平的升高或降低,进而引起小胶质细胞迁移和吞噬能力的增强或减弱。硒蛋白SelK有可能通过对脑部小胶质细胞吞噬和迁移能力的影响而对阿尔茨海默病的发生与否发挥作用。
[Abstract]:Alzheimer's disease (AD), also known as Alzheimer's disease, is a major disease that harms human health and quality of life, and affects the family and society. Microglia are macrophages in the brain, have immune activity, play the role of endogenous immune defense and protect neurons. In the early stage of Alzheimer's disease, the decrease of phagocytose and migration ability of microglia can not remove the deposited A tau protein in time, which is considered to be one of the important causes of Alzheimer's disease, so it is of great significance to improve the swallowing and migration ability of microglia. selenium is expressed in the form of selenium protein in human and animal life individuals, the selenium protein SelK is one of the high-content selenium proteins in the brain, It has been found that selenium protein SelK is capable of promoting the release of calcium ions in the ER by promoting the overexpression of IP3R, which is one of the main receptors to control calcium release on the ER, so as to improve the ion content in the cytoplasm. Whether selenium protein mSSelK and selenium can affect the migration and phagocytizing ability of microglia is an important problem. Five adenoviral vectors, such as mouse selenium protein mSSelK (mouse selenocarrK) overexpressing adenovirus vector, gene knock-down adenovirus vector and negative control, blank adenovirus vector control, were constructed. MTT assay was used to detect the effect of 5 adenovirus vectors on BV2 activity in resting and LPS-activated microglial cells. The results showed that 5 adenovirus vectors had no significant effect on the survival rate of resting and activated microglia, and the next experiment could be carried out. The effects of five adenovirus vectors on BV2 migration ability were investigated by Transwell chamber method and scratch-healing method. The results showed that both the Transwell chamber experiment and the scratch-healing experiment showed that the overexpression of selenium protein mSSelK/ gene knockdown could significantly increase/ decrease the migration ability of oligodendrocytes in resting state and activated state, and the migration ability might be increased or decreased to 180%/ 40%. The effect of 5 kinds of adenovirus vector on the devouring ability of mouse microglial cells (BV2) was detected by phagocytizing chicken blood red blood cells. The results showed that the overexpression of selenium protein mSSelK decreased the ability of resting state and LPS-activated microglial cells to phagocytize chicken blood red blood cells, and increased or decreased to 180%, 5% and 200%, 3%, respectively. In conclusion, selenium protein mSSelK plays an important role in microglial cells, and the increase of mSSelK expression of selenoprotein significantly increases the phagocytose and migration ability of microglial cells, while the reduction in the expression of selenium protein mSSelK significantly decreases the phagocyte and migration ability of microglial cells. The effect of 5 adenovirus vectors on the activation status of small glial cells (BV-2) was investigated by Griess method (detection of NO release). The results showed that only LPS activated the activation of microglial cells increased or decreased with the overexpression of selenium protein. It is indicated that only the increase/ decrease of phagocytose and migration ability of activated microglial cells is due to the change of the activation state of microglial cells. However, the change of phagocyte and migration ability of microglia in resting state did not affect the activation status of microglia due to the change of mSSelK expression. Secondly, flow cytometry was used to study the effect of the overexpression of selenium protein mSSelK on the level of free Ca 2 + in BV2 cytoplasm. It was found that the overexpression of selenium protein mSSelK caused the level of free Ca 2 + in cytoplasm to be increased to 300%, and the knockdown of selenium protein mSSelK caused the decrease of free Ca 2 + level to 60% in cytoplasm. In addition, qPCR and Western blot were used to investigate the effect of selenium protein mSSelK on the expression of IP3R (IP3R) in the control of calcium channel on ER. It has been found that the overexpression of mSSelK causes an increase in the expression of IP3R gene in cells, regardless of resting or activated microglia, whereas the knockdown of mSSelK causes a reduction in the expression of IP3R gene in cells. Finally, the effect of sodium selenite on microglial cells and the relationship between the expression of selenium protein and mSSelK were studied. MTT assay showed that sodium selenite, which was lower than or equal to 3. m u.M, had no inhibition on the growth of cell BV-2, which could inhibit cell growth or cause cell death. The effect of sodium selenite on cell activation was detected by Griess method. The results showed that sodium selenite had no significant effect on the activation status of microglia in resting state. Using real-time quantitative PCR and Western blot, the expression of mSSelK increased with the increase of sodium selenite, and there was dose-dependent effect. The results indicated that the change of trace element selenium could increase the expression of selenium protein mSSelK in mouse microglial cells. The increase or decrease of the expression level of selenium protein mSSelK in mouse microglial cells increases or decreases the expression of calcium ion channel protein IP3R on the ER, resulting in an increase or decrease in the level of bicarbonate ions in the cell cytoplasm, which in turn causes enhancement or reduction in the migration and swallowing capacity of microglial cells. The selenium protein SelK has the potential to play a role in the onset or absence of Alzheimer's disease by affecting the ability to phagocytize and migrate small glial cells in the brain.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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