本文選題：AQP4　切入點(diǎn)：3%氯化鈉　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景水通道蛋白(Aquaporins, AQPs)是一組與水的跨膜轉(zhuǎn)運(yùn)有關(guān)的膜蛋白家族,其中水通道蛋白4(Aquaporin-4, AQP4)在腦組織中廣泛分布,已有研究表明AQP4是星形膠質(zhì)細(xì)胞水腫形成的限速環(huán)節(jié)。本課題組前期的研究已發(fā)現(xiàn)用3%氯化鈉處理15min,星形膠質(zhì)細(xì)胞膜表面AQP4水平明顯降低,而20%甘露醇卻沒有此作用。提示3%氯化鈉減輕腦水腫、降低顱內(nèi)壓的機(jī)制除同20%甘露醇所具有的滲透性脫水機(jī)制外還有更重要的非滲透機(jī)制的參與。 目的觀察3%氯化鈉對細(xì)胞膜AQP4蛋白內(nèi)化的影響。 方法(1)采用pEGFP-AQP4-M23重組質(zhì)粒轉(zhuǎn)染U251細(xì)胞,建立穩(wěn)定轉(zhuǎn)染細(xì)胞系,通過抗AQP4免疫熒光法鑒定穩(wěn)定表達(dá)AQP4的U251細(xì)胞。(2)采用乳酸脫氫酶及MTT比色法觀察3%氯化鈉對U251細(xì)胞的損傷。(3)通過多功能酶標(biāo)儀、激光共聚焦及流式細(xì)胞儀三種方法觀察3%氯化鈉溶液對U251細(xì)胞AQP4內(nèi)化的影響。(4)通過多功能酶標(biāo)儀測定3%氯化鈉溶液對U251胞內(nèi)鈣離子濃度的影響。 結(jié)果1.成功構(gòu)建穩(wěn)定表達(dá)AQP4的U251細(xì)胞。2.20%甘露醇處理0-30min,細(xì)胞培養(yǎng)液中LDH含量及MTT細(xì)胞存活率均無明顯變化；3%氯化鈉處理0～25min,細(xì)胞培養(yǎng)液中LDH含量及MTT細(xì)胞存活率均無明顯變化,當(dāng)時間延長至30min時,雖細(xì)胞培養(yǎng)上清液中LDH含量無明顯增加,但細(xì)胞存活率與0min相比顯著下降(p0.05)。 3.20%甘露醇處理0～25min,U251細(xì)胞AQP4綠色熒光及細(xì)胞膜上抗AQP4抗體紅色熒光強(qiáng)度無明顯變化。3%氯化鈉處理0～25min對U251細(xì)胞AQP4綠色熒光強(qiáng)度無明顯影響,但在處理5min時即可顯著減少細(xì)胞膜抗AQP4抗體的紅色熒光強(qiáng)度(p0.05),隨處理時間延長至15min時細(xì)胞紅色熒光強(qiáng)度降到最低點(diǎn)；隨后隨著時間的延長至20min和25min細(xì)胞的紅色熒光強(qiáng)度有反彈上升；激光共聚焦顯微鏡下觀察到處理前U251細(xì)胞的綠色熒光主要集中表達(dá)在胞膜,3%氯化鈉處理后綠色熒光主要集中在胞漿中；流式細(xì)胞儀測定結(jié)果顯示,3%氯化鈉處理前后U251細(xì)胞AQP4的綠色熒光強(qiáng)度無明顯變化,而U251細(xì)胞膜上的抗AQP4抗體的紅色熒光及結(jié)合有抗體的陽性細(xì)胞比率明顯下降p0.05)。4.3%氯化鈉處理U251細(xì)胞15min可引起細(xì)胞內(nèi)鈣離子濃度的升高(p0.05)。 結(jié)論(1)成功建立穩(wěn)定表達(dá)AQP4綠色融合蛋白的U251細(xì)胞。(2)3%氯化鈉溶液處理U251細(xì)胞25min內(nèi)不引起細(xì)胞膜的損傷和細(xì)胞的死亡。(3)3%氯化鈉可促進(jìn)細(xì)胞膜上AQP4蛋白的內(nèi)化,20%甘露醇無此作用。(4)3%氯化鈉可提高細(xì)胞內(nèi)鈣離子濃度。
[Abstract]:Background Aquaporins (AQPs) is a family of membrane proteins associated with water transmembrane transport, in which aquaporin-4 (AQP4) is widely distributed in brain tissue. It has been shown that AQP4 is the rate-limiting link of astrocyte edema formation. Our previous study has found that the AQP4 level of astrocyte membrane surface decreased significantly after treated with 3% sodium chloride for 15 min. However, 20% mannitol had no such effect, suggesting that 3% sodium chloride can relieve brain edema and decrease intracranial pressure. Besides the osmotic dehydration mechanism of 20% mannitol, the mechanism of 3% sodium chloride is more important than that of osmotic dehydration. Objective to observe the effect of 3% sodium chloride on the internalization of AQP4 protein. Methods 1) U251 cells were transfected with pEGFP-AQP4-M23 recombinant plasmid, and stable transfected cell lines were established. The stable expression of AQP4 in U251 cells was identified by anti AQP4 immunofluorescence assay.) lactate dehydrogenase and MTT colorimetry were used to observe the damage of 3% sodium chloride to U251 cells. The effect of 3% sodium chloride solution on the AQP4 internalization of U251 cells was observed by laser confocal and flow cytometry. Results 1. When U251 cells with stable expression of AQP4 were successfully constructed with 2.20% mannitol for 0-30 min, there was no significant change in the content of LDH and the survival rate of MTT cells treated with 3% sodium chloride for 0 ~ 25 min. There was no significant change in the content of LDH and the survival rate of MTT cells in the cell culture medium. When the time was extended to 30 min, the cell survival rate decreased significantly compared with 0 min, although the content of LDH in the supernatant of cell culture did not increase significantly. The green fluorescence intensity of AQP4 in U251 cells treated with 3.20% mannitol for 25 min and the red fluorescence intensity of anti AQP4 antibody on cell membrane were not significantly changed. The green fluorescence intensity of AQP4 in U251 cells treated with 3% sodium chloride for 0 ~ 25 min had no obvious effect on the green fluorescence intensity of U251 cells. However, the red fluorescence intensity of cell membrane anti-#en0# antibody was significantly decreased after treatment for 5 minutes, and the red fluorescence intensity reached the lowest point when the treatment time was extended to 15 minutes. Then the red fluorescence intensity of the cells rebounded and increased with the prolongation of time to 20min and 25min. Laser confocal microscopy showed that the green fluorescence of U251 cells was mainly expressed in the cytoplasm after treated with 3% sodium chloride on the membrane. The results of flow cytometry showed that the green fluorescence intensity of AQP4 in U251 cells did not change significantly before and after 3% sodium chloride treatment. On the other hand, the red fluorescence of anti-#en0# antibody on U251 cell membrane and the ratio of positive cells bound to AQP4 antibody decreased significantly (p 0.05N 路4.3wt% NaCl) treatment for 15 min could induce the increase of intracellular Ca ~ (2 +) concentration in U251 cells (p _ (0.05)). Conclusion (1) the stable expression of AQP4 green fusion protein was successfully established in U251 cells treated with 3% sodium chloride solution for 25 minutes without cell membrane damage and cell death. 3% sodium chloride could promote the internalization of AQP4 protein on U251 cell membrane by 20% glycerol. 3% sodium chloride could increase the intracellular calcium concentration.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R725.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 孟淑珍,辛穎,韓曉華,韓玉昆,薛辛東;水通道蛋白4表達(dá)減少與新生大鼠缺氧缺血腦水腫的關(guān)系[J];中國當(dāng)代兒科雜志;2005年04期

2 李喬俊,岳少杰,虞佩蘭,尹飛,楊于嘉;高滲鈉液治療小兒感染性顱內(nèi)高壓癥26例分析[J];醫(yī)學(xué)臨床研究;2005年08期

3 付雪梅,姚裕家,楊釗,向龍,高舉;AQP4在新生大鼠實(shí)驗(yàn)性星形膠質(zhì)細(xì)胞缺氧損傷模型中的表達(dá)變化及意義[J];四川大學(xué)學(xué)報(醫(yī)學(xué)版);2005年05期

4 唐宇平;蔡定芳;劉軍;李文偉;陳依萍;聞名;;急性腦出血血腫周圍組織水通道蛋白-4表達(dá)與血腦屏障損傷的關(guān)系[J];中華神經(jīng)科雜志;2005年11期

5 陳英輝;趙永波;劉文文;咼登俊;王乃東;馬愛梅;;藥物難治性癲癇患者腦內(nèi)水通道蛋白-4的表達(dá)上調(diào)(英文)[J];Neuroscience Bulletin;2005年06期

，

本文編號：1569975