本文關(guān)鍵詞： 腺病毒 血色素結(jié)合蛋白 共培養(yǎng) 氧化損傷 腦出血　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：近年來大量研究證實顱內(nèi)出血后血腫裂解出來的亞鐵血紅素的氧化形式氯高鐵血紅素的蓄積對神經(jīng)細胞有直接的毒性作用，血色素結(jié)合蛋白能夠通過降低亞鐵血紅素的蓄積并加速其分解代謝。在我們的前期研究中發(fā)現(xiàn)，HPX基因敲除的老鼠腦出血后神經(jīng)功能的損傷較正常鼠嚴重，本文旨在探討血色素結(jié)合蛋白的高表達，在腦出血后繼發(fā)性腦損傷中對神經(jīng)元的保護作用。 方法：新生Sprague-Dawley(SD)乳鼠（1-3天），取皮層腦組織培養(yǎng)神經(jīng)細胞。構(gòu)建攜帶血色素結(jié)合蛋白基因的腺病毒感染神經(jīng)細胞（HPX組）24小時后，放入transwell小室與50ul動脈血進行共培養(yǎng);空白對照組（Blank組）與含28ul血清的50ulDMEM/F12共培養(yǎng)；模型組（Model組）神經(jīng)細胞感染空載腺病毒。分別取共培養(yǎng)后12小時與24小時細胞上清液和蛋白進行檢測。通過硫代巴比妥酸法檢測上清液中丙二醛（MDA）濃度,WST-1法檢測神經(jīng)細胞超氧化物歧化酶（SOD）活力，二硫代二硝基苯甲酸法檢測谷胱甘肽（GSH）含量。細胞凋亡試劑盒（流式細胞術(shù)）檢測細胞凋亡百分比以及免疫印跡法（WesternBlot）測定血紅素氧化酶1(HO-1)和細胞凋亡蛋白酶-3(caspase-3)表達量的變化。 結(jié)果：在血液與神經(jīng)細胞共培養(yǎng)12小時及24小時，模型組與HPX組相比，MDA在HPX組較模型組均有所下降（共培養(yǎng)12小時：4.05±0.89mmol/ml vs2.89±0.20mmol/ml P0.01；共培養(yǎng)24小時：4.22±0.96mmol/ml vs2.20±0.61mmol/ml; P0.05)。神經(jīng)細胞中總SOD活力在12小時及24小時，HPX組均較模型組有所增加，12小時：[41.32±6.39U/mgprot vs33.70±3.89U/mgpro (P0.05)]，24小時：[24.83±6.14U/mgpro vs10.62±2.37U/mgpro(P0.001)]�？侴SH含量在HPX組較模型組12小時及24小時均較有所增加：12小時：[P0.001;377.91±62.86mol/gpro vs253.13±63.18mol/gpro]，，24小時：[222.40±21.44mol/gpro vs152.50±16.47mol/gpro (P0.01)]。神經(jīng)細胞凋亡在共培養(yǎng)12小時時HPX組與模型組沒有明顯差異（P＞0.05），在24小時，細胞凋亡百分比在HPX組有所下降（P＜0.01）。Western法檢測HO-1的表達在24小時時HPX組表達下降（P＜0.01），Caspase-3的表達在兩個時間點HPX組較模型組均有所降低（Po.o1,P0.05）。 結(jié)論：通過增加血紅素結(jié)合蛋白的表達，可能血紅素結(jié)合蛋白通過更多的結(jié)合并轉(zhuǎn)運游離血紅素，對暴露于血液中的神經(jīng)細胞起到抗氧化損傷，保護腦出血后神經(jīng)細胞繼發(fā)性損傷的作用。
[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 鹵0.89 mmol / ml vs2.89 鹵0.20 mmol / ml P0.01; 24 h: 4.22 鹵0.96 mmol / ml vs2.20 鹵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 鹵6.39Ugprot vs33.70 鹵3.89Ugprot vs33.70 鹵3.89Ugpro P0.05] 24 hours: [24.83 鹵6.14Ugpro vs10.62 鹵2.37Ugpro vs10.62 鹵2.37Ugpro vs10.62 鹵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 鹵62.86 mol / g pro vs253.13 鹵63.18 mol / g pro] 24 hours: [222.40 鹵21.44 mol / g pro vs152.50 鹵16.47 mol / g pro vs152.50 鹵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).
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.34

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