甲基苯丙胺对神经细胞的毒性及烟酰胺的保护作用

发布时间：2018-04-21 09:53

本文选题：甲基苯丙胺 + 凋亡　；参考：《第一军医大学》2007年硕士论文

【摘要】： 甲基苯丙胺(Methamphetamine，METH)属于苯丙胺类兴奋剂(Amphetaminetype stimulants，ATS)，因其原料外观为纯白结晶体，晶莹剔透，故被吸毒、贩毒者称为“冰”(Ice)。由于它的毒性剧烈，人们便称之为“冰毒”。它具有药物依赖性(主要是精神依赖性)、中枢神经兴奋、致幻、食欲抑制和拟交感效应等药理、毒理学特性，是联合国精神药品公约管制的精神活性物质。由于该毒品可一次成瘾，其商品名称为SPEED(快速丸)。甲基苯丙胺可造成精神偏执，行为举止咄咄逼人，并引发反社会及性暴力倾向，还可引起吸服者失眠、幻觉、情绪低落，，同时严重损害内脏器官和脑组织，严重时导致肾机能衰竭及精神失常，甚至造成死亡。近年来，甲基苯丙胺在世界上不少国家和地区已成为主流毒品，制贩甲基苯丙胺的犯罪活动正日益猖獗。在国内，其滥用现象也在不断蔓延扩大。 METH对中枢神经系统的损害在哺乳动物体内的实验已经进行了广泛的研究，但是其确切的作用机制还没有完全的清楚。本实验通过体外海马细胞培养模型来研究METH的作用机制，去除动物体内的干扰因素，更加利于实验的控制，可以更加系统和直接的研究其对海马神经细胞的毒性作用，为METH的研究提供一条新的思路，从而更好的了解METH的作用机制。方法：用终浓度分别为0、0.8、1.6、3.2、6.4、12.8mmol／L的METH处理海马神经元48h，用MTT比色法分析细胞存活率，倒置显微镜和电镜观察凋亡的形态学改变，用流式细胞仪(FCM)检测细胞凋亡百分率。结果：METH可剂量依赖性的降低神经元的存活率，当METH浓度为6.4mmol／L时，其存活率为45.9％；倒置显微镜观察可见神经元胞体固缩，突起断裂，网络消失；电镜可见其细胞核明显固缩和凝聚；用流式细胞仪检查可见在0.8、1.6、3.2、6.4、12.8mmol／L METH处理海马神经元，细胞的凋亡率分别为(8.250±1.702)％、(19.525±2.495)％、(32.500±2.028)％、(49.975±1.603)％、(75.150±2.689)％，与对照组相比(3.150±0.603)％均有均显著性差异(P=0.000＜0.01)，且随剂量增加其凋亡百分率逐渐增大。(P=0.000＜0.01)。结论：一定浓度的甲基苯丙胺对体外培养的海马神经细胞具有细胞毒性，并且诱导神经细胞死亡具有剂量相关效应。烟酰胺，又名：维生素B3，是一种必须的营养物质，存在于谷物和家畜的肉中，常被用来作为一种营养补充剂。烟酰胺是烟酰胺腺嘌呤二核苷酸(辅酶I-NAD)的前体，并在线粒体电子传递链传递电子至氧，并释放能量，氧化磷酸化作用利用这些能量制造三磷酸腺苷(ATP)。参与体内葡萄糖酵解、脂肪代谢、丙酮酸代谢等过程，为脂质代谢，组织呼吸的氧化作用和糖元分解所需之成分。最近，对烟酰胺的研究表明，烟酰胺不仅是一种机体必须的营养物质，而且对细胞有着保护作用。对于烟酰胺的细胞保护作用，在越来越多的实验中得到了证实。Suman K. Mukherjee在小鼠的帕金森病模型中发现通过给C57BL／6的腹腔注射烟酰胺(500 mg／kg)，能够抑制脑的神经细胞的凋亡；并且注射烟酰胺8个小时后，发现脑的中脑区域的NAD水平从0.28±0.08 umol／g上升到0.38±0.04umol／g，在脑的纹状体区域，NAD的水平从0.35±0.04umol／g上升到0.45±0.04 umol／g，并且NAD的水平在注射烟酰胺13h后仍然保持着高水平。其它的研究显示对于神经细胞，烟酰胺在氧化应激、暂时性脑缺血、永久性局灶性脑缺血等动物模型中能够防止神经细胞的变性，并且还能防止内皮细胞的损伤，影响血管的生理学。本实验通过建立METH给药模型，通过注射烟酰胺，探讨烟酰胺对METH给药模型的保护作用及其可能的保护机制，对于METH中毒机制的研究具有一定的启发作用，为进一步探索METH治疗和干预提供一定的基础。方法：用高效液相色谱检测海马区和纹状体区DA含量；用NO、SOD、MDA试剂盒检测海马区和纹状体区中NO、SOD、MDA的含量变化；用原位缺口末端标记法(TUNEL)观察细胞凋亡情况及烟酰胺干预的影响。结果：METH给药模型中多巴胺和SOD的含量有显著性下降，预先应用烟酰胺能使多巴胺和SOD的下降有显著性减轻，烟酰胺+METH组与METH组间比较差异有显著性意义(P=0.000＜0.01)；METH给药模型中NO和MDA均有显著性上升，预先应用烟酰胺能使NO和MDA的下降有显著性减轻，烟酰胺+METH组与METH组间比较差异有显著性意义(P=0.000＜0.01)；METH给药模型中海马区和纹状体区可见大量的凋亡细胞，预先应用烟酰胺能使凋亡细胞显著性减少，烟酰胺+METH组与METH组间比较差异有显著性意义(P=0.000＜0.01)。结论：烟酰胺可以减轻METH对脑中DA系统和SOD的损伤，清除NO、MDA等自由基，对METH导致的大鼠脑神经细胞凋亡有明显的抑制作用，具有神经保护效应。
[Abstract]:Methamphetamine (METH) belongs to Amphetaminetype stimulants (ATS). Because its raw material is pure white crystalline and crystal clear, it is drug addicts and drug dealers call "Ice". Because of its toxicity, people call it "ice poison". It has drug dependence (mainly mental dependence). The central nervous excitement, phantom, appetite suppression, and quasi sympathetic effects, such as pharmacological, toxicological properties, are the psychoactive substances controlled by the United Nations Convention on psychotropic drugs. As the drug can be addicted to one time, the name of the drug is SPEED (rapid pill). Methamphetamine can cause mental obsession, aggressive behavior, and cause antisocial and sexual violence. Propensity can also cause insomnia, hallucination, low mood, and serious damage to viscera and brain tissue, which seriously cause renal failure and mental disorders, and even cause death. In recent years, methamphetamine has become the mainstream drug in many countries and regions in the world, and the crime of making methamphetamine is increasingly rampant. At home, its abuse is spreading.
The effects of METH on the damage of the central nervous system in mammals have been extensively studied, but the exact mechanism of the action has not been fully understood. In this experiment, the mechanism of METH was studied by the model of cultured hippocampal cells in vitro, the interference factors in animals were removed, and the control of the experiment could be more favorable. The systematic and direct study of its toxic effects on hippocampal neurons provides a new way of thinking for the study of METH, so as to better understand the mechanism of METH's action. Methods: the 48h of hippocampal neurons was treated with METH with final concentration of 0,0.8,1.6,3.2,6.4,12.8mmol / L, and the cell survival rate was analyzed by MTT colorimetry, inverted microscope and electron microscopy. Morphological changes of apoptosis were observed and the percentage of apoptosis was detected by flow cytometry (FCM). Results: METH could reduce the survival rate of neurons in a dose-dependent manner. When the concentration of METH was 6.4mmol / L, the survival rate was 45.9%. The nuclear fusion and condensation were obvious. The hippocampal neurons were treated with 0.8,1.6,3.2,6.4,12.8mmol / L METH by flow cytometry. The apoptosis rate of the cells was (8.250 + 1.702)%, (19.525 + 2.495)%, (32.500 + 2.028)%, (49.975 + 1.603)%, (75.150 + 2.689)%, respectively, (P=0) (P=0). .000 < 0.01), and the percentage of apoptosis increased gradually with the increase of dose. (P=0.000 < 0.01). Conclusion: a certain concentration of methamphetamine has cytotoxicity to cultured hippocampal neurons in vitro, and the induction of neural cell death has a dose-dependent effect.
Nicotinamide, also known as: vitamin B3, is a necessary nutrient that exists in the meat of grain and livestock and is often used as a nutritional supplement. Nicotinamide is the precursor of nicotinamide adenine dinucleotide (coenzyme I-NAD), and transmits electricity to oxygen in the mitochondrial electron transfer chain and releases energy, and is used for the acidification of phosphoric acid. Energy produces adenosine triphosphate (ATP). It participates in the process of glycolysis, fat metabolism, pyruvate metabolism and other processes, which are required for lipid metabolism, tissue respiration oxidation and glycogen decomposition.
Recently, studies on nicotinamide have shown that nicotinamide is not only a necessary nutrient for the body, but also a protective effect on cells. For the protective effect of nicotinamide, more and more experiments have shown that.Suman K. Mukherjee was found in the Parkinson's disease model of mice by intraperitoneal injection of C57BL / 6. Amines (500 mg / kg) can inhibit the apoptosis of brain neurons, and after 8 hours of injection of nicotinamide, the level of NAD in the brain region of the brain rises from 0.28 + 0.08 umol / g to 0.38 + 0.04umol / G. In the striatum region of the brain, the level of NAD rises from 0.35 + 0.04umol / g to 0.45 + 0.04 umol / G. Amides remain high after 13h. Other studies have shown that nicotinamide can prevent degeneration of nerve cells in animal models such as oxidative stress, temporary cerebral ischemia, permanent focal cerebral ischemia and other animal models, and also prevent endothelial cell damage and affect the physiology of blood vessels.
In this experiment, by establishing the METH drug delivery model, by injecting nicotinamide, the protective effect of nicotinamide on the METH drug delivery model and its possible protective mechanism are discussed. It has a certain inspiration for the study of the mechanism of METH poisoning and provides a certain basis for further exploration of the treatment and intervention of METH. Method: high performance liquid chromatography is used to detect the hippocampus. DA content in the region and striatum; the changes in the content of NO, SOD and MDA in the hippocampus and striatum were detected with NO, SOD and MDA kits. The apoptosis of cells and the effect of nicotinamide intervention were observed by in situ nick end labeling (TUNEL). Results: the content of dopamine and SOD in the METH administration model decreased significantly, and nicotinamide could be used in advance. The decrease of dopamine and SOD was significantly reduced, the difference of nicotinamide +METH group and METH group was significant (P=0.000 < 0.01); NO and MDA in the METH administration model increased significantly. The decrease of NO and MDA was significantly reduced in pre application of nicotinamide, and there was significant difference between the nicotinamide + METH group and the METH group (P=0.). 000 < 0.01); a large number of apoptotic cells were found in the CNOOC and striatum of the METH drug delivery model. Pre application of nicotinamide could reduce the significant number of apoptotic cells, and there was a significant difference between the nicotinamide +METH group and the METH group (P=0.000 < 0.01). Conclusion: nicotinamide can reduce the damage of METH to the DA system and SOD in the brain, remove NO, MDA and so on. Free radicals can obviously inhibit the apoptosis of rat brain neurons induced by METH, and have neuroprotective effects.

【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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