KCN慢性毒性及神经毒性机制研究

发布时间：2018-01-16 10:45

本文关键词：KCN慢性毒性及神经毒性机制研究　出处：《第二军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分KCN慢性毒性研究目的：观察KCN连续灌胃对Wistar大鼠的慢性毒性反应及其对各组织器官的损害程度、主要毒性靶器官及损害的可逆性，找到慢性毒性反应的有效剂量，为慢性氰化物中毒提供数据。方法：健康Wistar大鼠60只按体重随机分为对照组、低剂量组和高剂量组三组，每组20只，雌雄各半，按0mg/kg、5mg/kg、10mg/kg连续灌胃给药14天，恢复期7天。每天测定代谢笼中动物的食量，在给药第7天、第14天、恢复期第7天测定动物的体重。在给药第14天（d14，停药期）和第21天（d21，恢复期）每组各1/2大鼠采血测定红细胞计数、血红蛋白定量、红细胞压积、红细胞平均容积、红细胞平均血红蛋白容量、红细胞平均血红蛋白浓度、血小板计数、白细胞计数、中性粒细胞、淋巴细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞、大未染色细胞、网织红细胞计数、凝血酶原时间、活化部分凝血活酶时间、凝血酶时间及血浆纤维蛋白原等血液学指标以及血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶、碱性磷酸酶、乳酸脱氢酶、总胆红素、尿素氮、肌酐、总蛋白、白蛋白、血糖、总胆固醇、甘油三脂、肌酸磷酸激酶，血清钙、磷、血清钾、钠和氯等血液生化指标。在给药第14天和第21天分别对每组各1/2的动物剖检，进行组织病理学检查及骨髓细胞计数分类。结果：（1）给药组大鼠在给药后出现呼吸抑制、震颤等毒性反应，高剂量组较为严重，但均在20-30分钟内恢复正常。（2）连续灌胃KCN14天对大鼠的体重没有明显影响。（3）给药后各组动物的进食量没有明显变化。（4）KCN对大鼠的血液学指标没有明显影响。（5）KCN对大鼠的血液生化指标无明显影响。（6）KCN给药后，对大鼠的骨髓细胞没有明显影响。（7）停药期时，高剂量组雌性大鼠脑组织的绝对重量及相对重量均显著下降；部分高剂量组大鼠大脑皮质层小胶质细胞灶性增生，形成胶质小结样病变；灰白质交界处个别神经元细胞变性坏死，神经胶质细胞灶性浸润填充；更有个别动物大脑白质呈灶状空泡样变性，伴随有髓纤维数量的减少；部分动物甚至出现大脑基底部白质的灶性脱髓鞘样改变；少量动物海马回神经细胞锐减，甚至出现局灶性海马神经细胞大量脱落，且脱落区域可见大量神经胶质细胞浸润填充。其余脏器未见明显异常。恢复期时未见明显异常。结论：对大鼠连续14天的灌胃KCN试验，，给药组大鼠在给药时出现严重的毒性反应，毒性靶器官为中枢神经系统，毒性作用可逆。第二部分KCN神经毒性机制研究目的：结合KCN14天的灌胃试验，对Wistar大鼠大脑组织进行全基因组变化研究，找到KCN神经毒性反应可能涉及的信号通路；并对大鼠的血液和尿液进行代谢组学分析，揭示KCN给药对代谢产物的影响。方法：（1）基因组学研究：取12只大鼠（停药期0mg/kg剂量组4只，停药期10mg/kg剂量组4只，恢复期10mg/kg剂量组4只）的大脑组织，提取RNA，标记并扩增后与基因表达谱芯片进行杂交，经扫描分析筛选出给药组与对照组表达差异的基因。（2）代谢组学分析：收集对照组、低剂量组和高剂量组大鼠(每组8只)给药第14天的尿液及血清样本，做代谢组学分析。筛选出能体现KCN毒性的生物标志物。结果：（1）芯片杂交信号清晰，背景低，信噪比高，基因芯片检测和聚类分析结果有效。（2）经过T-test检验，筛选出显著上调（信号比值≥2）或显著下调（信号比值"f0.5）的差异表达基因289个。（3）按照基因功能分类体系（Gene Ontology, GO）分析差异表达基因，结果显示，差异基因体现的生物学功能多样，包括正向调节成神经细胞的增殖、大脑皮层发育、负向调节神经细胞凋亡、正向调节细胞增殖、钙离子应答过程、动作电位的调节、细胞分化的调节等。（4）导致KCN神经毒性的可能信号通路有：粘着斑信号通路、多巴胺能突触信号通路、谷氨酸能突触信号通路、细胞外基质受体相互作用信号通路、神经受体配体相互作用信号通路、神经营养因子信号通路、钙信号通路。（5）代谢组学分析：大鼠血清中磷酸胆碱，甘油磷酸胆碱，谷氨酰胺及甲基组氨酸的水平显著升高，赖氨酸的水平显著降低；大鼠尿液中氧化三甲胺，对羟基乙酸苯酯，羟丙茶碱，马尿酸盐，二甲胺水平相对溶媒对照组显著增加，丁二酸，乳酸盐，肌酸，瓜氨酸，乙酸盐水平显著降低。结论：（1）KCN的中枢神经毒性作用可能和其以下特性有关：KCN神经毒性的产生与神经细胞内钙稳态失调，Ca2+内流有关；KCN对粘着斑信号通路和细胞外基质受体相互作用信号通路的ITGB6有一定的调控作用，影响细胞与细胞间的相互作用；KCN对多巴胺能突触和谷氨酸能突触信号通路中GRIN2A有一定的调控作用，影响神经细胞间的突触传递和信号转导；对神经营养因子信号通路中NTRK2基因有一定的调控作用，影响神经营养因子与酪氨酸激酶受体Trk的结合，进而影响神经细胞的功能。（2）KCN神经毒性的产生，推测是有多条信号通路异常引起。其中最重要的机制可能是由于钙稳态失调，引发Ca2+内流，导致生物膜脂质过氧化，影响神经细胞的突触传递和信号转导，产生CNS毒性。（3）大鼠代谢组学研究结果提示，血清谷氨酰胺、赖氨酸及尿液中柠檬酸盐、丁二酸、乙酸盐水平变化可以作为KCN神经毒性的生物标志物，但需要进一步的研究证实。
[Abstract]:Part one chronic toxicity study of KCN
Objective: To observe the chronic toxicity of KCN continuous gastric Wistar of rats and on the tissue and organ damage, reversible main target organ toxicity and damage, find the effective dosage of chronic toxicity, provide data for chronic cyanide poisoning. Methods: 60 healthy Wistar rats were randomly divided into the control group, low dose group and high dose group three groups, 20 rats in each group, male and female, according to 0mg/kg, 5mg/kg, 10mg/kg continuous intragastric administration for 14 days, the recovery period for 7 days. Every animal in determination of metabolic cage intake in Administration for seventh days, fourteenth days, seventh days of rehabilitation by the weight of the animal. In the administration of fourteenth days (D14, withdrawal period) and twenty-first days (D21 recovery) 1/2 rats in each group were tested by red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin volume, red cell hemoglobin concentration in blood Plate count, leukocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, prothrombin time, reticulocyte count, activated partial thromboplastin time, thrombin time and fibrinogen and other hematological indexes and serum alanine aminotransferase. Aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, total protein, albumin, blood glucose, total cholesterol, glycerin three greases, creatine kinase, serum calcium, phosphorus, serum potassium, blood biochemical indexes of sodium and chloride. In the administration of fourteenth days and twenty-first days respectively for each group the 1/2 animal necropsy for histological examination and bone marrow cell count and pathological classification. Results: (1) to after the administration of respiratory medicine group rats were inhibited, tremor and other toxic reactions, the high dose group is more serious, but in 20-30 Minutes to return to normal. (2) by gavage for KCN14 days on the body weight of rats had no obvious effect. (3) to the food intake of each animal after the drug had no obvious changes. (4) no obvious effect of KCN on rat blood indexes. (5) the blood biochemical indexes in rats without KCN significant impact. (6) after administration of KCN on rat bone marrow cells had no obvious effect. (7) the withdrawal period, the absolute weight of brain tissue in female rats in high dose and relative weight were significantly decreased; part of the high dose group rat cortex microglial cells with focal hyperplasia the formation of glial nodule like lesions; gray white matter junction of individual neuronal cell degeneration and necrosis, glial cells infiltrated in filling; more individual animal cerebral white matter showed focal vacuolar degeneration, accompanied by reduced number of myelinated fibers; some animal or even basal brain focal demyelination like white matter changes A small animal; hippocampus nerve cells decreased, and even the emergence of a large number of off focal hippocampal neurons, and the shedding area shows a large number of glial cell infiltration filling. While other organs had no obvious abnormalities. The recovery stage had no obvious abnormalities. Conclusion: the rats for 14 consecutive days of intragastric administration group KCN test, rats severe toxicity in drug, the toxic target organs of the central nervous system toxicity was reversible.
The study of the second part KCN neurotoxicity mechanism
Objective: the gastric irrigation test of KCN14 days, the whole genome changes on brain tissue of rats with Wistar, find the KCN signaling pathway may be involved in neurotoxicity; and on rat blood and urine metabonomics analysis, reveal the effect of KCN administration on the metabolism. Methods: (1) genomics research: 12 rats (withdrawal period 0mg/kg dose group 4, withdrawal period 10mg/kg dose group 4, recovery 10mg/kg dose group 4 rats) of brain tissue, RNA extraction, amplification and labeling and gene expression microarray hybridization by scanning analysis to selected genes and drug group the control group (2). The expression of metabonomics analysis: collect control group, low dose group and high dose group rats (n = 8) for urine and serum samples of drug fourteenth days, do metabolomics analysis. Selected biomarkers can reflect the toxicity of KCN. Results: (1) hybrid chip Make clear signal, low background and high signal-to-noise ratio, gene chip detection and clustering analysis results. (2) after T-test test, screening was significantly up-regulated (signal ratio = 2) or decreased (the ratio of signal "f0.5) the 289 differentially expressed genes. (3) according to gene function classification system (Gene Ontology GO), gene expression, difference analysis results show that the biological function of genetic differences reflect the diversity, including the positive regulation of neural cell proliferation, cerebral cortex, negatively regulates neuronal apoptosis, positive regulation of cell proliferation, calcium ion response, regulation of action potential, cell differentiation and regulation of lead (4). The possible signal pathway of KCN neurotoxicity: focal adhesion pathway, dopaminergic synaptic pathways, glutamatergic synaptic signaling, extracellular matrix receptor interaction pathway, nerve receptor ligand interaction signal Lu, neurotrophic factor signaling pathway, calcium signaling pathway. (5) metabonomics analysis: choline phosphate in rat serum, glycerophosphocholine, glutamine and histidine methyl had significantly higher levels of lysine levels significantly decreased in rat urine; three oxidation of amine, hydroxyl phenyl acetate, proxyphyline two, hippurate, methylamine level relative to the vehicle control group increased significantly, succinic acid, lactate, creatine, citrulline, acetate decreased. Conclusion: (1) the central neurotoxicity of KCN may be related to the following characteristics: generation and nerve cells of KCN neurotoxicity in calcium homeostasis, Ca2+ influx; KCN the focal adhesion pathway and extracellular matrix receptor interaction pathway ITGB6 roles, interactions between cell and cell; KCN on dopaminergic synapses and glutamatergic synapses In the GRIN2A signaling pathway has a role in regulating the effects between nerve cells and synaptic transmission and signal transduction; has some effects on the regulation of NTRK2 signaling pathway in neurotrophic factor gene binding neurotrophic factor and tyrosine kinase receptor Trk, thereby affecting the nerve cell function. (2) KCN neurotoxicity, that is caused by abnormal multiple signaling pathways. One of the most important mechanism may be due to imbalance of calcium homeostasis, lead to Ca2+ influx, resulting in biological membrane lipid peroxidation, influence neuronal synaptic transmission and signal transduction, CNS toxicity. (3) rat Metabonomics Study results suggest that serum glutamine, lysine ammonia acid and urine citrate, succinate, acetate change level could be used as biomarkers of KCN neurotoxicity, but further research is needed to confirm that.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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