【作者】 潘彩飞；

【导师】 祝胜美；

【作者基本信息】 浙江大学 ， 麻醉学， 2010， 硕士

【摘要】 目的:研究异丙酚对氯化铵处理的大鼠脑皮质星形胶质细胞水通道蛋白-4(AQP-4)表达和细胞水肿的影响以及相关的作用机制。方法:体外培养Sprague Dawley大鼠脑皮质星形胶质细胞。待细胞培养3-4周左右时,用细胞免疫荧光标记星形胶质细胞特异性蛋白神经胶质酸性蛋白,星形胶质细胞纯度达95%以上的细胞备用。实验分组第一步:正常对照组、氯化铵(5mM)处理星形胶质细胞6h、12h、24h、48h组。取AQP-4表达明显升高的一个时间点24h作为药物处理后检测AQP-4的时间点。第二步:正常对照组、氯化铵(5mM)处理星形胶质细胞0.5h、6h、12h、24h组。取p-p38表达明显升高的时间点0.5h作为药物处理后检测p-p38的时间点。第三步:正常对照组、氯化铵(5mM)组、溶剂(DMSO)对照组、p38特异性抑制剂SB203580(10μM)组、异丙酚(10μM)组。其中溶剂、p38特异性抑制剂和异丙酚均分别与氯化铵共培养星形胶质细胞。用细胞免疫荧光法检测培养的星形胶质细胞纯度;用光学显微镜观察各组细胞形态,用蛋白免疫印迹法(western-blot)检测AQP-4、p38和p-p38的表达情况。结果:1、培养的星形胶质细胞的纯度为96.6%±1.4%。2、氯化铵处理星形胶质细胞12h后,AQP-4蛋白表达较正常对照组明显增加(P＜0.01),一直持续到48h。3、氯化铵处理星形胶质细胞后p38蛋白表达无明显变化;p-p38蛋白表达在氯化铵处理0.5h后较正常对照组明显增高(P＜0.05),在作用24h后p-p38表达仍明显升高。4、光学显微镜观察发现氯化铵处理的星形胶质细胞较正常组细胞肿胀明显,异丙酚和p38抑制剂能减轻氯化铵引起的细胞肿胀程度。异丙酚和p38抑制剂处理组星形胶质细胞AQP-4和p-p38蛋白表达较氯化铵处理组降低(P＜0.05)。p38蛋白表达在各组之间无明显统计学差异(P=0.341)。结论:异丙酚能抑制氯化铵弓j起的星形胶质细胞AQP-4的表达上调,减轻细胞的肿胀程度,其作用可能与抑制p38信号途径激活有关。更多还原

【Abstract】 Background: Astrocyte swelling is an important consequence of hepatic encephalopathy, and aquaporin-4 （AQP-4） has been reported to play vital role in this swelling. Ammonia causes astrocyte swelling and is also known to modulate AQP-4 expression in the astrocyte foot. Propofol is used to control intracranial pressure by reduce cerebral edema. The purpose of this article was to investigate the effects of propofol on ammonia-induced astrocyte swelling and AQP-4 expression in rats and its possible mechanisms.Methods: Astrocytes were separated from newborn Sprague Dawley rats. After cultured for 3-4 weeks, fluorescent glial fibrillary acidic protein labeling was used to evaluate the purity of cultured astrocytes, which achieved to 95% was considered to be used in this study. Cultured astrocytes were randomly divided into several groups, step 1: normal control group, ammonia-incubated for 6h, 12h, 24h, 48h group. We selected the 24h as the point we detected the expression of aquaporin-4 after we treated with drugs below; step 2: normal control group, ammonia-incubated for 0.5h, 6h, 12h, 24h group. We selected the 0.5h as the point we detect the expression of p-p38 after we treated with drugs below; step 3: normal control group, ammonia-incubated for 24h group, dimethyl sulfoxide （DMSO） control group, p38 antagonist SB203580（10μM） treatment group, propofol（10μM） treatment group. The purity of cultured astrocytes was evaluated by fluorescent glial fibrillary acidic protein labeling; cell morphology was assessed by light microscopy; the expression of AQP-4, p38 and p-p38 were detected by westem-blot analysis.Results: The purity of cultured astrocytes was achieved to 96.6%±1.4%. Astrocytes swelled significantly when exposed to 5 mM NH₄Cl for 24 h as compared to non-exposed astrocytes. Co-treatment with SB203580 （an inhibitor of p38） or propofol attenuated the degree of NH₄Cl-induced astrocyte swelling. Western blot analysis revealed that the expression levels of phospho-p38 and AQP-4 in NH₄Cl-treated cells were significantly increased relative to the control group （P < 0.05）; SB203580 or propofol co-treatment inhibited the increased expression of p-p38 and AQP-4 relative to the NH₄Cl-treated group （P < 0.05）. There were no significant differences in total p38 expression among the groups （P =0.341）.Conclusions: Ammonia-induced over-expression of AQP4 in astrocytes is regulated by the p38 MAPK pathway. Propofol prevented ammonia-induced AQP-4 protein over-expression may through inhibiting p38 MAPK activation.更多还原