维生素C对异氟醚引起的细胞毒性及认知功能损伤的作用研究

发布时间：2018-01-17 12:26

本文关键词：维生素C对异氟醚引起的细胞毒性及认知功能损伤的作用研究　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：术后认知功能障碍（postoperative cognitive dysfunction, POCD）是临床手术后常见的中枢神经系统并发症，具体表现为在手术麻醉结束后一段时间内，患者出现记忆力受损、内心焦虑以及短时间性格改变等症状，在老年患者中较为常见。术后认知功能障碍对患者的术后生活影响较大，会导致患者精神焦躁、并发症加重、延长康复时间，甚至增加发展为患阿尔茨海默病（Alzheimer’s disease,AD）的几率。从目前的研究资料来看，与术后认知功能障碍相关的因素中，麻醉是除年龄外对患者记忆力影响最大的风险因素。麻醉药物的细胞毒性以及对中枢神经系统的抑制作用，可能是术后认知功能障碍的发病机制。同时，在发生术后认知功能障碍的临床案例中，老年患者占有较大比例。每年约有6600万年龄超过65岁的老年人需要接受麻醉手术。因此，探明麻醉药物的细胞毒性机制以及减缓麻醉药物带来的细胞毒性，保证患者，尤其是老年患者的麻醉安全，是当下神经生物学及麻醉医学的研究热点。本研究利用分子生物学和细胞生物学等方法，在课题组先前的研究基础上，选取人体生命活动必需的维生素C作为抗氧化药物，在细胞水平试验以及动物行为学试验中证明，维生素C可以缓解由麻醉药物异氟醚引起的细胞凋亡以及小鼠学习与记忆能力下降，具体包括以下四个方面研究内容：（1）通过细胞内活性氧簇含量测定试验发现，维生素C和异氟醚共处理的H4-AAP细胞与异氟醚处理组细胞相比活性氧簇的含量降低，说明维生素C可以为氧化自由基提供阳离子，减少活性氧簇的蓄积。（2）通过细胞线粒体通透性转换孔（mPTP）检测试验、线粒体膜电势染色试验、ATP测定等试验发现，，维生素C可以抑制由异氟醚引起的H4-AAP细胞线粒体通透性转换孔开放、线粒体膜电势降低以及ATP生成减少。说明维生素C可以改善异氟醚引起的线粒体损伤，保持线粒体膜结构完整，维持电化学梯度平衡。（3）利用免疫印迹、MTT等实验技术，检测细胞内凋亡标志物Caspase-3的激活程度和细存活率，发现维生素C可以减轻由异氟醚引起的Caspase-3激活，提高细胞存活率，从而抑制细胞凋亡的发生。（4）利用条件性恐惧实验模型，观察异氟醚麻醉处理后小鼠的记忆情况，发现小鼠注射维生素C后，可以改善异氟醚引起的小鼠对环境以及声音的“僵直时间”减少，表明维生素C可以减轻由异氟醚引起的小鼠学习与记忆能力下降。综上所述，维生素C可以缓解麻醉药物异氟醚引起的H4 APP细胞活性氧簇蓄积、保护线粒体完整性和抑制细胞凋亡。条件性恐惧试验结果表明在小鼠行为学模型中维生素C对异氟醚产生的学习与记忆能力下降具有明显的保护作用。本研究结果为由异氟醚引起的术后认知功能障碍的防治用药提供了理论依据和试验数据。
[Abstract]:Postoperative cognitive dysfunction (POCD) is a common complication of central nervous system after operation. The symptoms included memory impairment, anxiety and short-term personality change after anesthesia. It is more common in the elderly patients. Postoperative cognitive dysfunction has a greater impact on postoperative life, which will lead to mental anxiety, aggravated complications and prolonged rehabilitation time. It even increases the risk of developing Alzheimer's disease (AD). From the current research data, the factors related to postoperative cognitive dysfunction. Anesthesia is the most important risk factor for patients' memory in addition to age. The cytotoxicity of anesthetic drugs and the inhibition of central nervous system may be the pathogenesis of postoperative cognitive dysfunction. In the clinical cases of postoperative cognitive dysfunction, the elderly patients account for a large proportion. Each year, about 66 million people over 65 years of age need anesthesia surgery. It is a hot topic in neurobiology and anesthetic medicine to find out the mechanism of cytotoxicity of anesthetic drugs and to reduce the cytotoxicity of anesthetic drugs and to ensure the safety of anesthesia in patients especially in the elderly. In this study, molecular biology and cell biology methods were used to select vitamin C, which is necessary for human life activities, as antioxidant drugs based on previous research by our research group. In cell level tests and animal behavioral tests, vitamin C can alleviate apoptosis induced by the anesthetic isoflurane and the ability of learning and memory in mice. Including the following four aspects of research content: 1) through the determination of intracellular reactive oxygen species concentration test found. The content of reactive oxygen species in H4-AAP cells treated with vitamin C and isoflurane was lower than that in isoflurane group, indicating that vitamin C could provide cations for oxidation free radicals. Reducing the accumulation of reactive oxygen species (Ros) was detected by mitochondrial permeability transition pore (mPTP) and mitochondrial membrane potential staining (mPTP). Vitamin C can inhibit the opening of mitochondrial permeability transition pore in H4-AAP cells induced by isoflurane. The decrease of mitochondrial membrane potential and the decrease of ATP production indicate that vitamin C can improve mitochondrial damage induced by isoflurane and maintain the integrity of mitochondrial membrane structure. Maintain the electrochemical gradient balance. 3) the activation degree and fine survival rate of apoptosis marker Caspase-3 were detected by Western blotting and other experimental techniques. It was found that vitamin C could reduce the activation of Caspase-3 induced by isoflurane, improve cell survival, and thus inhibit the occurrence of apoptosis. The memory of mice treated with isoflurane anesthesia was observed. It was found that after injection of vitamin C, isoflurane could improve the reduction of environment and sound rigidity time of mice induced by isoflurane. It was suggested that vitamin C could reduce the ability of learning and memory induced by isoflurane in mice. In conclusion, vitamin C can alleviate the accumulation of reactive oxygen species in H4 APP cells induced by isoflurane. The results of conditioned fear test showed that vitamin C could protect the learning and memory ability of isoflurane in mice. The results provide theoretical basis and experimental data for the prevention and treatment of postoperative cognitive dysfunction caused by isoflurane.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614

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