创伤性颅脑损伤后肠道上皮细胞线粒体功能变化的实验研究

发布时间：2018-03-20 19:47

  本文选题：创伤性颅脑损伤　切入点：肠道　出处：《苏州大学》2014年博士论文　论文类型：学位论文

【摘要】：创伤性颅脑损伤（traumatic brain injury, TBI）是神经外科最常见的疾病，随着工业及交通运输业的发展，其发病率逐年升高。因为其高致死率和致残率，TBI一直以来都被临床医生和众多学者重视。而事实上，相当一部分TBI患者并非原发病致死，而是多器官功能障碍（multiple organ dysfunction syndrome, MODS）致死。许多研究表明，，肠道不仅是MODS的靶器官，更是MODS的启动者。鉴于此，人们越来越重视TBI后肠功能紊乱的情况，对TBI后肠道屏障功能的研究也成为当今医学领域的一个重要课题和热点。 TBI后常发生肠功能障碍。肠功能障碍主要表现为胃肠动力下降，肠黏膜营养吸收障碍，黏膜破坏等。已有的研究主要集中在肠黏膜的显微结构改变，黏膜屏障破坏，黏膜血流变化以及炎症反应等，确切的机制仍不清楚。为了保证重要器官如心脑的血供，TBI后肠黏膜上皮细胞常处于缺血或低灌注状态。不幸地是，肠黏膜上皮细胞能量储备有限，不足以应对缺血或低灌注的状态。所以，肠黏膜上皮细胞对缺血缺氧非常敏感。一旦TBI发生，肠黏膜上皮细胞将面对强烈的氧化应激。 线粒体作为机体氧化磷酸化和能量供应的"重要工厂"，在氧化应激中扮演了重要的角色。一方面，线粒体呼吸作用可以产生机体所需的ATP，同时，呼吸作用也会产生大量反应性氧族（reactive oxygen species, ROS），从而进一步发生氧化损伤。虽然线粒体与能量代谢密切相关，但尚未发现有关TBI后肠道黏膜上皮细胞线粒体功能变化的研究。 第一部分两步法制备高质量大鼠小肠上皮细胞线粒体 目的：探讨一种两步制备高质量大鼠小肠上皮细胞线粒体的方法。方法：首先，用胶原酶和透明质酸酶消化法制备高质量的大鼠小肠上皮细胞；第二步再参考从培养的组织细胞中提取线粒体的方法从第一步制备的大鼠小肠上皮细胞中提取线粒体。BCA蛋白定量试剂盒测定制备的线粒体蛋白浓度；Western-blotting检测样品及对照品中Pax-5、β-actin、Cox IV的浓度来反映制备的线粒体纯度及污染情况；测定制备的线粒体完整性、稳定性以及生物活性。结果：检测制备的20例大鼠小肠上皮细胞线粒体样本，最低浓度为2.2108μg/μl，最高浓度为4.4516μg/μl，平均浓度为3.1932μg/μl；制备的线粒体样品中Pax-5、β-actin表达量比较低，Cox IV表达量较高；以细胞色素C氧化酶为标志酶在0~4℃保温2h，检测线粒体完整性仍在97%以上；制备的线粒体中细胞色素C氧化酶能很好的氧化NADH和苹果酸，且其活性可以被1mmol/L KCN抑制；制备的线粒体ATP酶含量与对照组（从培养的IEC-6细胞株制备的线粒体）无明显差别。结论：两步法制备大鼠肠道上皮细胞线粒体方法简便，制备的线粒体蛋白浓度和纯度较高，胞质和细胞核污染少，线粒体完整性和稳定性较好，线粒体活性也保持地较好，适于线粒体功能实验研究。 第二部分创伤性颅脑损伤（TBI）后大鼠肠道上皮细胞线粒体呼吸功能及相关酶活性的变化 目的：观察创伤性颅脑损伤（TBI）后大鼠肠道上皮细胞线粒体呼吸功能及相关酶活性的变化情况。方法：雄性SD大鼠56只，随机分为7组（每组8只）,包括对照组、脑损伤后6、12、24h和2、3、7d各1组。采用Feeney自由落体撞击法制作TBI模型。使用Clark氧电极测定线粒体呼吸功能（呼吸控制率和ADP/O比）；线粒体呼吸链复合物I-IV以及相关酶（丙酮酸脱氢酶、α-酮戊二酸脱氢酶、苹果酸脱氢酶）活性用分光光度计测定。结果：与对照组相比，创伤性颅脑损伤后大鼠肠道上皮细胞线粒体呼吸控制率在伤后6h开始下降，并且一直持续到伤后7d仍低于对照组(control,5.42±0.46;6h,5.20±0.18;12h,4.55±0.35;24h,3.75±0.22;2d,4.12±0.53;3d,3.45±0.41;7d,5.23±0.24; P＜0.01)。磷氧比在伤后12h开始显著下降，一直持续到术后3d(12h,3.30±0.10;24h,2.61±0.21;2d,2.95±0.18;3d,2.76±0.09; P＜0.01)。伤后24h和3d各可以观察到1个峰值。线粒体呼吸链复合物I (6h:110±10,12h:115±12,24h:85±9,2d:80±15,3d:65±16; P＜0.01)和复合物II (6h:105±8,12h:110±92,24h:80±10,2d:76±8,3d:68±12; P＜0.01)的活性在伤后6h和12h先增高，然后从伤后24h开始下降，并持续到伤后3d。与对照组相比，线粒体呼吸链复合物III和复合物IV伤后变化无明显差别。此外，与对照组相比，丙酮酸脱氢酶活性在伤后6h开始下降，并且一直持续到伤后7d仍低于对照组(6h:90±8,12h:85±10,24h:65±12,2d:60±9,3d:55±6,7d:88±11; P＜0.01)。α-酮戊二酸脱氢酶活性变化规律与丙酮酸脱氢酶类似，但其活性下降是从伤后12h开始的(12h:90±12,24h:80±9,2d:76±15,3d:68±7,7d:90±13; P＜0.01)。苹果酸脱氢酶活性伤后无明显变化。结论：创伤性颅脑损伤后线粒体呼吸功能和部分相关酶活性收到抑制，线粒体功能不良可能在创伤性颅脑损伤后肠道功能障碍的发生中扮演重要角色。 第三部分创伤性颅脑损伤后大鼠肠道上皮细胞线粒体膜电位和通透性转换的改变 目的：观察创伤性颅脑损伤（TBI）后大鼠肠道上皮细胞线粒体膜电位和通透性转换的改变情况。方法：雄性SD大鼠96只，体重220～260g，按随机数字法分均为假手术对照组和TBI组，每组按照术后3、6、12h和1、3、7d分为6个亚组（n=8）：采用Feeney自由落体撞击法制作TBI模型，假手术组对照组仅行右侧顶部开窗而无TBI。分别于TBI后3、6、12h和1、3、7d处死各组8只大鼠，分离肠黏膜上皮细胞，用流式细胞检测细胞凋亡指数，成功提取肠道上皮细胞线粒体后分别测定线粒体膜电位（MMP）和通透性转换孔（MPTP）活性。结果：与假手术对照组比较，TBI组凋亡指数从术后6h起明显升高，一直持续到术后7d(P＜0.05），从术后3h起肠道上皮细胞线粒体MMP即明显降低(P＜0.05）而MPTP活性明显增高(P＜0.05）。结论：大鼠TBI后肠道上皮细胞凋亡率增加，肠道上皮细胞线粒体MMP及MPTP活性的改变可能在这一病理生理过程中发挥重要作用。
[Abstract]:Traumatic brain injury (traumatic brain, injury, TBI) is the most common disease in Department of Neurosurgery, along with the development of industry and transportation industry, its incidence increased year by year. Because of its high mortality rate and disability rate, TBI has always been clinicians and many scholars attention. In fact, quite a part of TBI patients is not the primary disease but death, multiple organ dysfunction (MODS multiple organ dysfunction syndrome) to death. Many studies show that intestinal tract is not only the target organ of MODS, MODS is a start. In view of this, people pay more and more attention to TBI functional bowel disorder situation, research on the intestinal barrier function after TBI has become an important issue and a hot field in modern medicine.
Intestinal dysfunction often occurs after TBI. Intestinal dysfunction mainly decreased gastrointestinal motility, intestinal malabsorption, mucosal damage. The change of microstructure research has been mainly concentrated in the intestinal mucosa, mucosal barrier damage, mucosal blood flow changes and inflammatory reaction, the exact mechanism is still not clear. In order to ensure the important organs such as heart the blood supply of TBI intestinal epithelial cells in ischemia or hypoperfusion. Unfortunately, intestinal epithelial cells and energy reserves is limited, not enough to cope with ischemia or low perfusion state. Therefore, the intestinal mucosal epithelial cells are very sensitive to ischemia and hypoxia. Once TBI occurs, the intestinal epithelial cells will face oxidative stress strong.
As the body of mitochondrial oxidative phosphorylation and energy supply of the "factory", plays an important role in oxidative stress. On the one hand, mitochondrial respiration can produce the body's ATP, at the same time, the respiration will produce large amounts of reactive oxygen species (reactive oxygen, species, ROS), so as to further oxidative damage although closely related to mitochondrial energy metabolism, but have not yet found the changes of mitochondrial function of intestinal mucosal epithelial cells of the TBI.
The first part of the two step method to prepare the mitochondria of small intestinal epithelial cells in high quality rats
Objective: To investigate the two step method for preparing high quality mitochondria of intestinal epithelial cells in rats. Methods: first, the production of high quality with collagenase and hyaluronidase digestion method of intestinal epithelial cells in rats; the second step according to the determination of the concentration of mitochondrial protein extract preparation of mitochondrial.BCA protein assaykit intestinal epithelial cells the extraction method of mitochondria from cultured cells from the first step of preparation in rats; Western-blotting samples and the control sample in Pax-5, beta -actin, IV concentration of Cox to reflect the purity of mitochondria and the pollution of preparation; Determination of mitochondrial integrity of preparation, stability and biological activity. Results: detection 20 cases of rat intestinal epithelial cell mitochondrial sample preparation, the lowest concentration of 2.2108 mu g/ Mu L, the highest concentration of 4.4516 mu g/ Mu L, the average concentration of 3.1932 mu g/ Mu L; mitochondrial sample preparation In Pax-5, beta -actin expression is relatively low, Cox IV high expression; the cytochrome C oxidase enzyme marked 2H insulation at 0~4 DEG C, detection of mitochondrial integrity is still more than 97%; the preparation of mitochondrial cytochrome C oxidase in good oxidation of NADH and malic acid, and its activity can be 1mmol/L KCN inhibition; preparation of mitochondrial ATP enzyme and the content of the control group (IEC-6 cells cultured from the mitochondrial preparation) had no significant difference. Conclusion: the two step preparation of mitochondria in intestinal epithelial cells in rats, simple preparation, high purity and concentration of mitochondrial protein, cytoplasmic and nuclear pollution, mitochondrial integrity and good stability, mitochondrial activity also maintained good, suitable for experimental study of mitochondrial function.
The changes of mitochondrial respiratory function and related enzyme activity in the intestinal epithelial cells of the second part of traumatic brain injury (TBI)
Objective: To observe the effect of traumatic brain injury (TBI) changes of mitochondrial respiratory function of intestinal epithelial cells of rats and related enzyme activity. Methods: 56 male SD rats were randomly divided into 7 groups (n = 8), including control group, 6,12,24h after brain injury and 1 2,3,7d group. The TBI model Feeney free fall impact method. Determination of mitochondrial respiratory function using Clark oxygen electrode (respiratory control ratio and ADP/O ratio); mitochondrial respiratory chain complex I-IV and related enzymes (pyruvate dehydrogenase, - ketoglutarate dehydrogenase, malate dehydrogenase) activity was measured by a spectrophotometer. Results: compared with control group, traumatic brain after the injury of mitochondria in rat intestinal epithelial cells of respiratory control rate at 6h after injury began to decline, and continued until 7d after injury is still lower than that of the control group (control, 5.42 + 0.46 + 0.18; 6h, 5.20; 12h, 4.55 + 0.35 + 0.22; 24h, 3.75; 2D, 4.12 + 0.5 3;3d,3.45卤0.41;7d,5.23卤0.24; P锛

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