腺病毒介导的HPX基因高表达对与血液共培养的神经细胞保护作用的实验研究

发布时间：2018-02-27 17:02

本文关键词： 腺病毒血色素结合蛋白共培养氧化损伤脑出血　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：近年来大量研究证实颅内出血后血肿裂解出来的亚铁血红素的氧化形式氯高铁血红素的蓄积对神经细胞有直接的毒性作用，血色素结合蛋白能够通过降低亚铁血红素的蓄积并加速其分解代谢。在我们的前期研究中发现，HPX基因敲除的老鼠脑出血后神经功能的损伤较正常鼠严重，本文旨在探讨血色素结合蛋白的高表达，在脑出血后继发性脑损伤中对神经元的保护作用。方法：新生Sprague-Dawley(SD)乳鼠（1-3天），取皮层脑组织培养神经细胞。构建携带血色素结合蛋白基因的腺病毒感染神经细胞（HPX组）24小时后，放入transwell小室与50ul动脉血进行共培养;空白对照组（Blank组）与含28ul血清的50ulDMEM/F12共培养；模型组（Model组）神经细胞感染空载腺病毒。分别取共培养后12小时与24小时细胞上清液和蛋白进行检测。通过硫代巴比妥酸法检测上清液中丙二醛（MDA）浓度,WST-1法检测神经细胞超氧化物歧化酶（SOD）活力，二硫代二硝基苯甲酸法检测谷胱甘肽（GSH）含量。细胞凋亡试剂盒（流式细胞术）检测细胞凋亡百分比以及免疫印迹法（WesternBlot）测定血红素氧化酶1(HO-1)和细胞凋亡蛋白酶-3(caspase-3)表达量的变化。结果：在血液与神经细胞共培养12小时及24小时，模型组与HPX组相比，MDA在HPX组较模型组均有所下降（共培养12小时：4.05±0.89mmol/ml vs2.89±0.20mmol/ml P0.01；共培养24小时：4.22±0.96mmol/ml vs2.20±0.61mmol/ml; P0.05)。神经细胞中总SOD活力在12小时及24小时，HPX组均较模型组有所增加，12小时：[41.32±6.39U/mgprot vs33.70±3.89U/mgpro (P0.05)]，24小时：[24.83±6.14U/mgpro vs10.62±2.37U/mgpro(P0.001)]。总GSH含量在HPX组较模型组12小时及24小时均较有所增加：12小时：[P0.001;377.91±62.86mol/gpro vs253.13±63.18mol/gpro]，，24小时：[222.40±21.44mol/gpro vs152.50±16.47mol/gpro (P0.01)]。神经细胞凋亡在共培养12小时时HPX组与模型组没有明显差异（P＞0.05），在24小时，细胞凋亡百分比在HPX组有所下降（P＜0.01）。Western法检测HO-1的表达在24小时时HPX组表达下降（P＜0.01），Caspase-3的表达在两个时间点HPX组较模型组均有所降低（Po.o1,P0.05）。结论：通过增加血红素结合蛋白的表达，可能血红素结合蛋白通过更多的结合并转运游离血红素，对暴露于血液中的神经细胞起到抗氧化损伤，保护脑出血后神经细胞继发性损伤的作用。
[Abstract]:Objective: in recent years, a large number of studies have proved that the accumulation of hemoglobin, the oxidized form of hemin, has a direct toxic effect on nerve cells after intracerebral hemorrhage. Hemochrome binding protein can reduce the accumulation of heme and accelerate its catabolism. In our previous study, we found that the brain function of mice with HPX gene knockout was more serious than that of normal mice after intracerebral hemorrhage. The purpose of this study was to investigate the high expression of hemoglobin binding protein (HBP) and its protective effect on neurons in the secondary brain injury after intracerebral hemorrhage (ICH). Methods: Neonatal Sprague-Dawley (SD) neonatal rats were cultured in cortical brain tissue for 1 to 3 days. After 24 hours of construction of HPX infected nerve cells with adenovirus containing hemoglobin binding protein gene, the cells were co-cultured in transwell chamber and 50ul arterial blood. The blank control group (Blank group) was co-cultured with 50ulDMEM / F12 containing 28ul serum. Model group (model group) neurons were infected with empty adenovirus. Cell supernatants and proteins were detected at 12 hours and 24 hours after co-culture. The concentration of malondialdehyde (MDAs) in supernatants was detected by thiobarbituric acid method and WST-1 method was used to detect the concentration of malondialdehyde (MDA) in the supernatants. Nerve cell superoxide dismutase (SOD) activity, The content of glutathione glutathione (GSH) was detected by dithiodinitrobenzoic acid assay, the percentage of cell apoptosis was detected by flow cytometry (FCM) and the expression of heme oxygenase 1 (HO-1) and apoptotic protease 3 (caspase-3) were detected by Western blot assay. Results: the blood and nerve cells were co-cultured for 12 hours and 24 hours. Compared with HPX group, HPX group was lower than model group (12 hours after incubation: 4.05 卤0.89 mmol / ml vs2.89 卤0.20 mmol / ml P0.01; 24 h: 4.22 卤0.96 mmol / ml vs2.20 卤0.61 mmol / ml; P0.05.The total SOD activity in nerve cells was increased at 12 hours and 24 hours after HPX compared with model group. H: [41.32 卤6.39Ugprot vs33.70 卤3.89Ugprot vs33.70 卤3.89Ugpro P0.05] 24 hours: [24.83 卤6.14Ugpro vs10.62 卤2.37Ugpro vs10.62 卤2.37Ugpro vs10.62 卤2.37Ugprot P 0.001]. Total GSH content in HPX group was significantly higher than that in model group for 12 hours and 24 hours: [P 0.001n 377.91 卤62.86 mol / g pro vs253.13 卤63.18 mol / g pro] 24 hours: [222.40 卤21.44 mol / g pro vs152.50 卤16.47 mol / g pro vs152.50 卤16.47 mol / g pro P 0.01]. There was no significant difference in apoptosis between HPX group and model group at 12 h, 24 h, 24 h. The percentage of apoptosis in the HPX group was decreased (P < 0.01). Western method was used to detect the expression of HO-1 in the HPX group at 24 hours. The expression of Caspase-3 in the HPX group was lower than that in the model group at two time points (P < 0.01). The expression of Caspase-3 in the HPX group was lower than that in the model group. Conclusion: by increasing the expression of heme binding protein, it is possible that heme binding protein may have antioxidant damage to nerve cells exposed to blood by more binding and transport of free heme. To protect the secondary injury of nerve cells after intracerebral hemorrhage (ICH).
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34

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