小鼠硒蛋白mSelK及硒對小膠質(zhì)細胞的作用及與阿爾茲海默病的關(guān)系研究
發(fā)布時間：2018-10-21 12:01
【摘要】：阿爾茨海默癥(Alzheimer's disease, AD)又稱老年癡呆癥,是一種危害人類身體健康和生命質(zhì)量,影響家庭和社會的重大疾病,隨著我國進入老齡化社會,相關(guān)研究更具有現(xiàn)實意義。小膠質(zhì)細胞是腦內(nèi)的巨噬細胞,具有免疫活性,發(fā)揮著內(nèi)源性免疫防御作用和保護神經(jīng)元等功能。在阿爾茨海默病的發(fā)生早期,小膠質(zhì)細胞吞噬和遷移能力的降低,不能及時清除沉積的Aβ蛋白,被認為是阿爾茨海默病發(fā)生的重要原因之一,因此提高小膠質(zhì)細胞的吞噬和遷移能力具有重要意義。硒在人類和動物生命個體中以硒蛋白的形式表達,硒蛋白SelK是腦中含量較高的硒蛋白之一,已有研究發(fā)現(xiàn)硒蛋白SelK能夠通過促進內(nèi)質(zhì)網(wǎng)上控制鈣釋放主要受體之一的IP3 Receptor (IP3R)的過表達促進內(nèi)質(zhì)網(wǎng)中儲存鈣離子的釋放,使胞漿中游離鈣離子含量的提高。硒蛋白mSelK和硒是否會影響小膠質(zhì)細胞的遷移和吞噬能力是急需解決的重要問題。首先構(gòu)建了小鼠硒蛋白mSelK ( mouse selenoprotein K )過表達的腺病毒載體及基因敲減腺病毒載體及其陰性對照,空白腺病毒載體對照等5種腺病毒載體。采用MTT法檢測5種腺病毒載體對靜息態(tài)和與LPS激活的小鼠小膠質(zhì)細胞BV2活力的影響。結(jié)果顯示5種腺病毒載體對靜息態(tài)和活化態(tài)小膠質(zhì)細胞的存活率均無顯著影響,可進行下一步實驗研究。采用Transwell小室法和劃痕愈合實驗法考察了 5種腺病毒載體對靜息態(tài)和LPS激活小鼠小膠質(zhì)細胞BV2遷移能力的影響。結(jié)果顯示,無論Transwell小室實驗還是劃痕愈合實驗均表明硒蛋白mSelK的過表達/基因敲減能夠顯著提高/降低靜息態(tài)和激活狀態(tài)小膠質(zhì)細胞的遷移能力,且遷移能力大概增高或降低到180%/40%。采用吞噬雞血紅細胞實驗法檢測了 5種腺病毒載體對小鼠小膠質(zhì)細胞BV2吞噬能力的影響。結(jié)果表明硒蛋白mSelK的過表達/基因敲減能使靜息態(tài)和LPS激活小膠質(zhì)細胞的吞噬雞血紅細胞能力顯著增加/降低,且吞噬和率吞噬指數(shù)分別增加或降低至為180%,5%和200%,3%。綜上所述,硒蛋白mSelK對小膠質(zhì)細胞有著重要的作用,硒蛋白mSelK表達的增加顯著提高小膠質(zhì)細胞的吞噬和遷移能力,而硒蛋白mSelK表達的減少顯著降低小膠質(zhì)細胞的吞噬和遷移能力。為探討硒蛋白mSelK表達對小膠質(zhì)細胞吞噬和遷移能力的影響是否是引起小膠質(zhì)細胞的活化而導(dǎo)致的,通過Griess法(檢測NO釋放)探討了 5種腺病毒載體對小鼠小膠質(zhì)細胞BV-2活化狀態(tài)的影響。實驗結(jié)果表明,在引起小膠質(zhì)細胞吞噬和遷移能力增加及降低的硒蛋白mSelK過表達/基因敲減腺病毒載體侵染滴度下,只有LPS激活小膠質(zhì)細胞的活化程度隨著硒蛋白的過表達/敲減而增加或減少,說明只有活化態(tài)小膠質(zhì)細胞吞噬和遷移能力的增加/減少是由于小膠質(zhì)細胞活化狀態(tài)的改變而導(dǎo)致的。而靜息態(tài)小膠質(zhì)細胞吞噬和遷移能力的改變并不是由于mSelK表達的變化影響到了小膠質(zhì)細胞的活化狀態(tài)。其次采用流式細胞儀方法研究了硒蛋白mSelK的過表達/基因敲減對BV2胞漿中游離Ca2+水平的影響。研究發(fā)現(xiàn),無論對于靜息態(tài)還是活化態(tài)的小膠質(zhì)細胞,硒蛋白mSelK的過表達均引起胞漿中游離Ca2+的水平的升高至300%;而硒蛋白mSelK的敲減引起胞漿中游離Ca2+水平的降低至60%。另外采用qPCR和Western Blot考察了硒蛋白mSelK對內(nèi)質(zhì)網(wǎng)上控制鈣離子通道開放關(guān)鍵蛋白酶IP3R (IP3 receptor)表達的影響。研究發(fā)現(xiàn)無論對于靜息態(tài)的還是活化態(tài)的小膠質(zhì)細胞,mSelK的過表達引起細胞內(nèi)IP3R基因表達的增多;而mSelK的敲減引起細胞內(nèi)IP3R基因表達的減少。最后研究了亞硒酸鈉對小膠質(zhì)細胞的影響及與硒蛋白mSelK表達之間的關(guān)系。MTT實驗結(jié)果顯示,對于靜息態(tài)小膠質(zhì)細胞BV-2來說,低于或等于3μM的亞硒酸鈉對其生長無抑制,超過此濃度就會抑制細胞生長或引起細胞死亡。采用Griess法檢測亞硒酸鈉作用于BV-2細胞對細胞活化狀態(tài)的影響。實驗結(jié)果表明,亞硒酸鈉對靜息態(tài)小膠質(zhì)細胞的活化狀態(tài)沒有顯著的影響。采用實時定量PCR和Western Blot證實硒蛋白mSelK的表達隨著亞硒酸鈉的增多而增多,且有劑量依賴的效應(yīng)。綜合以上結(jié)果可以得出如下結(jié)論:微量元素硒的變化可以增加小鼠小膠質(zhì)細胞中硒蛋白mSelK表達的變化。小鼠小膠質(zhì)細胞中硒蛋白mSelK的表達量的升高或降低通過增加或減少內(nèi)質(zhì)網(wǎng)上鈣離子通道蛋白IP3R的表達,從而引起細胞漿中游離鈣離子水平的升高或降低,進而引起小膠質(zhì)細胞遷移和吞噬能力的增強或減弱。硒蛋白SelK有可能通過對腦部小膠質(zhì)細胞吞噬和遷移能力的影響而對阿爾茨海默病的發(fā)生與否發(fā)揮作用。
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16
本文編號：2285033
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16
【參考文獻】
相關(guān)期刊論文 前10條
1 烏蘭其其格;高晶;周偉光;陳玉惠;敖特根巴雅爾;;IBRV對牛單核巨噬細胞吞噬雞紅細胞功能的影響[J];畜牧與飼料科學(xué);2015年04期
2 崔瑞娜;宋樂樂;陽幼榮;梁艷;趙衛(wèi)國;吳雪瓊;;結(jié)核菌感染巨噬細胞鈣離子通道表達的研究[J];中國病原生物學(xué)雜志;2014年11期
3 陳東亞;陸羅定;俞萍;卞倩;徐軍;楊明晶;;流式細胞術(shù)檢測小鼠腹腔巨噬細胞吞噬能力的方法學(xué)探討[J];中國免疫學(xué)雜志;2014年08期
4 劉紅梅;黃開勛;徐輝碧;;內(nèi)質(zhì)網(wǎng)硒蛋白的研究進展[J];中國科學(xué):化學(xué);2014年04期
5 顧曙余;梁魯曉雪;李蓮;張雪茜;李靜;曹祥榮;;“小鼠腹腔巨噬細胞吞噬實驗”方法的改進與研究[J];安徽農(nóng)學(xué)通報;2013年23期
6 余虹;;小鼠巨噬細胞吞噬功能測定方法的改進及應(yīng)用(英文)[J];生命科學(xué)研究;2013年04期
7 韋美丹;林繼宗;朱寧;王克萬;王勇;;Aβ_(1-42)作用的小膠質(zhì)細胞對體外培養(yǎng)的神經(jīng)干細胞生存的影響[J];中國病理生理雜志;2012年04期
8 田金可;吳秋玨;王恬;;硒的抗氧化功能及在動物生產(chǎn)中的應(yīng)用[J];中國飼料;2011年23期
9 湯偉;彭求賢;蔡紅兵;李欣;莫志賢;;丹參多糖對免疫抑制小鼠單核吞噬細胞吞噬功能的影響[J];時珍國醫(yī)國藥;2011年10期
10 黃月;任秀花;張善峰;張杰文;;不同劑量移植小膠質(zhì)細胞對阿爾茨海默病大鼠腦內(nèi)β-淀粉樣蛋白的作用[J];中華醫(yī)學(xué)雜志;2011年33期
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