本文選題：高滲鈉 + 水通道蛋白4　； 參考：《中南大學(xué)》2014年博士論文

【摘要】：背景水通道蛋白(Aquaporins,AQPs)是一組介導(dǎo)水分子跨膜轉(zhuǎn)運(yùn)的蛋白家族,在哺乳動(dòng)物體內(nèi)廣泛存在。AQP4是人體中樞神經(jīng)系統(tǒng)最主要的水通道蛋白,與腦水腫和顱高壓的發(fā)生發(fā)展密切相關(guān)。已有大量的研究表明,AQP4是細(xì)胞毒性腦水腫時(shí)星形膠質(zhì)細(xì)胞水腫的限速環(huán)節(jié)。高滲鈉液是另一種滲透性脫水劑,有研究表明高滲鈉液可以通過降低星形膠質(zhì)細(xì)胞上AQP4的表達(dá)來治療腦水腫及顱高壓。本課題組前期研究已經(jīng)發(fā)現(xiàn)3%氯化鈉直接快速處理原代星形膠質(zhì)細(xì)胞15min可使細(xì)胞膜表面AQP4含量減少,并進(jìn)一步發(fā)現(xiàn)3%氯化鈉所致星形膠質(zhì)瘤U251細(xì)胞膜上AQP4含量的減少是由于AQP4發(fā)生內(nèi)化的結(jié)果。 目的探討高滲鈉在細(xì)胞外培養(yǎng)基達(dá)到何種滲透濃度及處理時(shí)間時(shí)可引起體外培養(yǎng)的星形膠質(zhì)瘤U251細(xì)胞細(xì)胞膜上AQP4發(fā)生內(nèi)化,以及引起這種內(nèi)化發(fā)生是由鈉離子還是氯離子所介導(dǎo)。 方法1.采用pEGFP-AQP4-M23重組質(zhì)粒轉(zhuǎn)染U251細(xì)胞,通過倒置熒光顯微鏡觀察AQP4綠色融合蛋白的陽性表達(dá)。2.采用MTT比色法觀察不同處理方式對(duì)U251細(xì)胞的損傷。3.通過多功能酶標(biāo)儀確定高滲鈉加入細(xì)胞外培養(yǎng)基使培養(yǎng)基滲透濃度增加至何種濃度及處理多長(zhǎng)時(shí)間可引起U251細(xì)胞膜上AQP4內(nèi)化。使用不同的含鈉液及含氯液確定這種作用是由何種離子所介導(dǎo)。4.使用流式細(xì)胞術(shù)及激光共聚焦觀察的方法進(jìn)一步確定以上結(jié)論。 結(jié)果1.成功建立表達(dá)AQP4的U251細(xì)胞。2.在細(xì)胞外培養(yǎng)液滲透濃度升高至920mOsm/L時(shí)甘露醇對(duì)細(xì)胞的存活率無明顯影響,而高滲鈉在培養(yǎng)液滲透濃度達(dá)到820mOsm/L時(shí)即可引起細(xì)胞的存活率明顯下降(P0.05)。甘露醇組在不同甘露醇滲透濃度條件下對(duì)AQP4蛋白綠色熒光(反映U251細(xì)胞總AQP4蛋白含量)無明顯影響,對(duì)膜表面的抗AQP4-PE抗體的紅色熒光(反映U251細(xì)胞膜膜上AQP4含量)也沒有明顯的影響。而高滲鈉組在不同滲透濃度條件下雖對(duì)AQP4蛋白綠色熒光無明顯影響,但在氯化鈉滲透濃度達(dá)到400mOsm/L時(shí)細(xì)胞膜表面的紅色熒光出現(xiàn)了明顯的減少(P0.05),并隨滲透濃度的逐漸增大而進(jìn)一步減少(P0.01),至滲透濃度達(dá)到720mOsm/L后這種進(jìn)一步減少的現(xiàn)象消失。提示當(dāng)細(xì)胞外液氯化鈉透濃度達(dá)到720mOsm/L時(shí)是促進(jìn)AQP4內(nèi)化的最佳滲透濃度。3.在720mOsm/L甘露醇滲透濃度條件下甘露醇處理30min對(duì)細(xì)胞存活率無明顯影響,但720mOsm/L氯化鈉滲透濃度條件下高滲鈉組處理30min即對(duì)存活率有明顯影響,提示高滲鈉組處理時(shí)間小于30min時(shí)對(duì)細(xì)胞存活率無明顯影響。甘露醇組在720mOsm/L甘露醇滲透濃度條件下不同的處理時(shí)間對(duì)AQP4蛋白綠色熒光無明顯影響,對(duì)膜表面的紅色熒光也沒有明顯的影響(P0.05)。高滲鈉組在氯化鈉720mOsm/L滲透濃度條件下處理不同時(shí)間雖對(duì)AQP4蛋白綠色熒光無明顯影響,但在處理10min時(shí)細(xì)胞膜表面的紅色熒光出現(xiàn)了明顯的減少(P0.05),并隨處理時(shí)間的逐漸延長(zhǎng)而進(jìn)一步減少(P0.01),處理15min后這種進(jìn)一步減少情況達(dá)到最大,隨后處理時(shí)間延長(zhǎng),細(xì)胞膜表面的抗AQP4抗體的紅色熒光強(qiáng)度出現(xiàn)反彈上升。提示處理15min時(shí)是促進(jìn)AQP4蛋白內(nèi)化的最佳處理時(shí)間。4.不同的處理組對(duì)U251細(xì)胞內(nèi)AQP4綠色熒光均無明顯的影響。硝酸鈉組、硫氰化鈉組與對(duì)照組相比,其AQP4-PE抗體的紅色熒光強(qiáng)度無明顯的差異。但氯化鋰組和氯化膽堿組與對(duì)照組相比,其抗AQP4-PE抗體的紅色熒光強(qiáng)度明顯降低,且這種差異與氯化鈉組相差不大,提示高滲鈉促進(jìn)U251細(xì)胞膜表面AQP4蛋白內(nèi)化的作用是由氯離子介導(dǎo)的,而非鈉離子。進(jìn)一步的流式細(xì)胞術(shù)及激光共聚焦顯微鏡觀察均證實(shí)此結(jié)果。 結(jié)論1.成功建立表達(dá)AQP4的U251細(xì)胞。 2.高滲鈉可促進(jìn)星形膠質(zhì)瘤U251細(xì)胞膜上AQP4內(nèi)化,引起星形膠質(zhì)瘤U251細(xì)胞膜上AQP4內(nèi)化的最佳氯化鈉滲透濃度為720mOsm/L,最佳作用時(shí)間為15min。 3.高滲鈉促進(jìn)U251細(xì)胞膜上AQP4內(nèi)化是由氯離子所介導(dǎo),而非鈉離子。
[Abstract]:Background Aquaporins (AQPs) is a group of proteins that mediate the transmembrane transport of water molecules..AQP4 is the most important aquaporin in the human central nervous system in mammals, which is closely related to the development of brain edema and intracranial hypertension. A large amount of research has shown that AQP4 is the star of cytotoxic brain edema. The speed limit of glial cell edema. Hypertonic sodium is another osmotic dehydrating agent. Studies have shown that hypertonic sodium can be used to treat brain edema and cranial pressure by reducing the expression of AQP4 on astrocytes. A preliminary study in our group has found that 3% sodium chloride is directly located in the glial cell 15min to make the cell membrane The content of AQP4 on the surface decreased, and it was further found that the decrease of AQP4 content on U251 cell membrane caused by 3% NaCl was due to internalization of AQP4.
Objective to investigate the osmotic concentration and treatment time of hypertonic sodium in the extracellular medium, which can induce the internalization of AQP4 on the cell membrane of Astroglioma U251 cells in vitro, and cause this internalization to be mediated by sodium ions or chloride ions.
Method 1. U251 cells were transfected by pEGFP-AQP4-M23 recombinant plasmid, and the positive expression of AQP4 green fusion protein was observed by inverted fluorescence microscope. The MTT colorimetric method was used to observe the damage to U251 cells by MTT colorimetric method, and.3. was determined by the multi-function enzyme labeling method to determine the infiltration concentration of hypertonic sodium to the extracellular medium. Concentration and treatment for how long can cause AQP4 internalization on the membrane of U251 cells. Using different sodium containing liquid and chlorine containing liquid to determine which ion mediated.4. using flow cytometry and laser confocal observation to further determine the above conclusion.
Results 1. the successful establishment of AQP4 U251 cell.2. had no significant effect on the survival rate of mannitol when the infiltration concentration of extracellular medium increased to 920mOsm/L, and the survival rate of cells decreased significantly (P0.05) when the permeability of hypertonic sodium reached 820mOsm/L (P0.05). The mannitol group was in the conditions of different mannitol infiltration concentration. There was no obvious effect on the green fluorescence of AQP4 protein (the total AQP4 protein content of U251 cells). The red fluorescence of the anti AQP4-PE antibody on the membrane surface (reflecting the AQP4 content on the membrane membrane of U251 cells) was also not significantly affected. While the hypertonic sodium group had no obvious effect on the green fluorescence of the AQP4 protein at different osmotic concentrations, but in the sodium chloride permeation. When the concentration reached 400mOsm/L, the red fluorescence of the surface of the cell membrane decreased significantly (P0.05), and decreased with the increasing of the concentration of osmotic concentration (P0.01), and the further decrease disappeared after the concentration of osmosis reached 720mOsm/L. It was suggested that when the concentration of sodium chloride in the extracellular fluid reached 720mOsm/L, it was the most important to promote the internalization of AQP4. Under the condition of 720mOsm/L mannitol infiltration concentration of.3., 30min had no obvious effect on cell survival, but the treatment of 30min in hypertonic sodium group under the concentration of 720mOsm/L NaCl concentration had a significant effect on the survival rate, suggesting that the treatment time of hypertonic sodium group was less than 30min had no obvious effect on cell survival. Under the condition of 720mOsm/L mannitol permeation concentration, the different treatment time had no obvious effect on the green fluorescence of AQP4 protein, but had no obvious effect on the red fluorescence of the membrane surface (P0.05). The treatment of sodium chloride group at different time of sodium chloride 720mOsm/L permeation concentration had no obvious effect on the green fluorescence of AQP4 protein, but in the treatment of 10min. The red fluorescence of the surface of the cell membrane decreased significantly (P0.05) and decreased with the gradual extension of the treatment time (P0.01). The further reduction reached the maximum after the treatment of 15min, and then the treatment time was prolonged. The red fluorescence intensity of the anti AQP4 antibody on the cell membrane surface was bounced up. It was suggested that the treatment of 15min was the case. The best treatment time to promote the internalization of AQP4 protein.4. had no obvious effect on the AQP4 green fluorescence in U251 cells. The sodium thiocyanate group had no significant difference in red fluorescence intensity of the AQP4-PE antibody compared with the control group, but the lithium chloride group and the choline chloride group were compared with the control group, and their anti AQP4-PE antibodies were red. The intensity of color fluorescence was significantly reduced, and the difference was not much different from that in the sodium chloride group. It was suggested that the effect of hypertonic sodium on the internalization of AQP4 protein on the membrane surface of U251 cells was mediated by chloride ion, but not by sodium ions.
Conclusion 1. U251 cells expressing AQP4 were successfully established.
2. hypertonic sodium can promote the internalization of AQP4 on the membrane of astrocytoma U251 cells. The optimum concentration of sodium chloride permeation is 720mOsm/L in the AQP4 internalization of Astroglioma U251 cell membrane, and the best time is 15min..
3. hypertonic sodium promotes the internalization of AQP4 on U251 cell membrane by chloride ion rather than sodium ion.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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2 李敏;陳少軍;陳學(xué)群;杜繼曾;;腦水腫的AQP4調(diào)節(jié)機(jī)制研究進(jìn)展[J];浙江大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2013年01期

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本文編號(hào)：1820909