3%氯化钠促进U251细胞膜AQP4蛋白内化的研究

发布时间：2018-03-05 11:05

本文选题：AQP4　切入点：3%氯化钠　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景水通道蛋白(Aquaporins, AQPs)是一组与水的跨膜转运有关的膜蛋白家族,其中水通道蛋白4(Aquaporin-4, AQP4)在脑组织中广泛分布,已有研究表明AQP4是星形胶质细胞水肿形成的限速环节。本课题组前期的研究已发现用3%氯化钠处理15min,星形胶质细胞膜表面AQP4水平明显降低,而20%甘露醇却没有此作用。提示3%氯化钠减轻脑水肿、降低颅内压的机制除同20%甘露醇所具有的渗透性脱水机制外还有更重要的非渗透机制的参与。目的观察3%氯化钠对细胞膜AQP4蛋白内化的影响。方法(1)采用pEGFP-AQP4-M23重组质粒转染U251细胞,建立稳定转染细胞系,通过抗AQP4免疫荧光法鉴定稳定表达AQP4的U251细胞。(2)采用乳酸脱氢酶及MTT比色法观察3%氯化钠对U251细胞的损伤。(3)通过多功能酶标仪、激光共聚焦及流式细胞仪三种方法观察3%氯化钠溶液对U251细胞AQP4内化的影响。(4)通过多功能酶标仪测定3%氯化钠溶液对U251胞内钙离子浓度的影响。结果1.成功构建稳定表达AQP4的U251细胞。2.20%甘露醇处理0-30min,细胞培养液中LDH含量及MTT细胞存活率均无明显变化；3%氯化钠处理0～25min,细胞培养液中LDH含量及MTT细胞存活率均无明显变化,当时间延长至30min时,虽细胞培养上清液中LDH含量无明显增加,但细胞存活率与0min相比显著下降(p0.05)。 3.20%甘露醇处理0～25min,U251细胞AQP4绿色荧光及细胞膜上抗AQP4抗体红色荧光强度无明显变化。3%氯化钠处理0～25min对U251细胞AQP4绿色荧光强度无明显影响,但在处理5min时即可显著减少细胞膜抗AQP4抗体的红色荧光强度(p0.05),随处理时间延长至15min时细胞红色荧光强度降到最低点；随后随着时间的延长至20min和25min细胞的红色荧光强度有反弹上升；激光共聚焦显微镜下观察到处理前U251细胞的绿色荧光主要集中表达在胞膜,3%氯化钠处理后绿色荧光主要集中在胞浆中；流式细胞仪测定结果显示,3%氯化钠处理前后U251细胞AQP4的绿色荧光强度无明显变化,而U251细胞膜上的抗AQP4抗体的红色荧光及结合有抗体的阳性细胞比率明显下降p0.05)。4.3%氯化钠处理U251细胞15min可引起细胞内钙离子浓度的升高(p0.05)。结论(1)成功建立稳定表达AQP4绿色融合蛋白的U251细胞。(2)3%氯化钠溶液处理U251细胞25min内不引起细胞膜的损伤和细胞的死亡。(3)3%氯化钠可促进细胞膜上AQP4蛋白的内化,20%甘露醇无此作用。(4)3%氯化钠可提高细胞内钙离子浓度。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

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