高渗钠对体外培养星形胶质瘤U251细胞膜AQP4内化的影响研究

发布时间：2018-04-29 17:35

本文选题：高渗钠 + 水通道蛋白4　；参考：《中南大学》2014年博士论文

【摘要】：背景水通道蛋白(Aquaporins,AQPs)是一组介导水分子跨膜转运的蛋白家族,在哺乳动物体内广泛存在。AQP4是人体中枢神经系统最主要的水通道蛋白,与脑水肿和颅高压的发生发展密切相关。已有大量的研究表明,AQP4是细胞毒性脑水肿时星形胶质细胞水肿的限速环节。高渗钠液是另一种渗透性脱水剂,有研究表明高渗钠液可以通过降低星形胶质细胞上AQP4的表达来治疗脑水肿及颅高压。本课题组前期研究已经发现3%氯化钠直接快速处理原代星形胶质细胞15min可使细胞膜表面AQP4含量减少,并进一步发现3%氯化钠所致星形胶质瘤U251细胞膜上AQP4含量的减少是由于AQP4发生内化的结果。目的探讨高渗钠在细胞外培养基达到何种渗透浓度及处理时间时可引起体外培养的星形胶质瘤U251细胞细胞膜上AQP4发生内化,以及引起这种内化发生是由钠离子还是氯离子所介导。方法1.采用pEGFP-AQP4-M23重组质粒转染U251细胞,通过倒置荧光显微镜观察AQP4绿色融合蛋白的阳性表达。2.采用MTT比色法观察不同处理方式对U251细胞的损伤。3.通过多功能酶标仪确定高渗钠加入细胞外培养基使培养基渗透浓度增加至何种浓度及处理多长时间可引起U251细胞膜上AQP4内化。使用不同的含钠液及含氯液确定这种作用是由何种离子所介导。4.使用流式细胞术及激光共聚焦观察的方法进一步确定以上结论。结果1.成功建立表达AQP4的U251细胞。2.在细胞外培养液渗透浓度升高至920mOsm/L时甘露醇对细胞的存活率无明显影响,而高渗钠在培养液渗透浓度达到820mOsm/L时即可引起细胞的存活率明显下降(P0.05)。甘露醇组在不同甘露醇渗透浓度条件下对AQP4蛋白绿色荧光(反映U251细胞总AQP4蛋白含量)无明显影响,对膜表面的抗AQP4-PE抗体的红色荧光(反映U251细胞膜膜上AQP4含量)也没有明显的影响。而高渗钠组在不同渗透浓度条件下虽对AQP4蛋白绿色荧光无明显影响,但在氯化钠渗透浓度达到400mOsm/L时细胞膜表面的红色荧光出现了明显的减少(P0.05),并随渗透浓度的逐渐增大而进一步减少(P0.01),至渗透浓度达到720mOsm/L后这种进一步减少的现象消失。提示当细胞外液氯化钠透浓度达到720mOsm/L时是促进AQP4内化的最佳渗透浓度。3.在720mOsm/L甘露醇渗透浓度条件下甘露醇处理30min对细胞存活率无明显影响,但720mOsm/L氯化钠渗透浓度条件下高渗钠组处理30min即对存活率有明显影响,提示高渗钠组处理时间小于30min时对细胞存活率无明显影响。甘露醇组在720mOsm/L甘露醇渗透浓度条件下不同的处理时间对AQP4蛋白绿色荧光无明显影响,对膜表面的红色荧光也没有明显的影响(P0.05)。高渗钠组在氯化钠720mOsm/L渗透浓度条件下处理不同时间虽对AQP4蛋白绿色荧光无明显影响,但在处理10min时细胞膜表面的红色荧光出现了明显的减少(P0.05),并随处理时间的逐渐延长而进一步减少(P0.01),处理15min后这种进一步减少情况达到最大,随后处理时间延长,细胞膜表面的抗AQP4抗体的红色荧光强度出现反弹上升。提示处理15min时是促进AQP4蛋白内化的最佳处理时间。4.不同的处理组对U251细胞内AQP4绿色荧光均无明显的影响。硝酸钠组、硫氰化钠组与对照组相比,其AQP4-PE抗体的红色荧光强度无明显的差异。但氯化锂组和氯化胆碱组与对照组相比,其抗AQP4-PE抗体的红色荧光强度明显降低,且这种差异与氯化钠组相差不大,提示高渗钠促进U251细胞膜表面AQP4蛋白内化的作用是由氯离子介导的,而非钠离子。进一步的流式细胞术及激光共聚焦显微镜观察均证实此结果。结论1.成功建立表达AQP4的U251细胞。 2.高渗钠可促进星形胶质瘤U251细胞膜上AQP4内化,引起星形胶质瘤U251细胞膜上AQP4内化的最佳氯化钠渗透浓度为720mOsm/L,最佳作用时间为15min。 3.高渗钠促进U251细胞膜上AQP4内化是由氯离子所介导,而非钠离子。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【参考文献】