本文關鍵詞：KCN慢性毒性及神經毒性機制研究　出處：《第二軍醫(yī)大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： KCN 毒性靶器官 慢性毒性 神經毒性 毒性機制 基因芯片 神經毒性 信號通路 代謝組學

【摘要】：第一部分KCN慢性毒性研究 目的：觀察KCN連續(xù)灌胃對Wistar大鼠的慢性毒性反應及其對各組織器官的損害程度、主要毒性靶器官及損害的可逆性，找到慢性毒性反應的有效劑量，為慢性氰化物中毒提供數據。方法：健康Wistar大鼠60只按體重隨機分為對照組、低劑量組和高劑量組三組，每組20只，雌雄各半，按0mg/kg、5mg/kg、10mg/kg連續(xù)灌胃給藥14天，恢復期7天。每天測定代謝籠中動物的食量，在給藥第7天、第14天、恢復期第7天測定動物的體重。在給藥第14天（d14，停藥期）和第21天（d21，恢復期）每組各1/2大鼠采血測定紅細胞計數、血紅蛋白定量、紅細胞壓積、紅細胞平均容積、紅細胞平均血紅蛋白容量、紅細胞平均血紅蛋白濃度、血小板計數、白細胞計數、中性粒細胞、淋巴細胞、單核細胞、嗜酸性粒細胞、嗜堿性粒細胞、大未染色細胞、網織紅細胞計數、凝血酶原時間、活化部分凝血活酶時間、凝血酶時間及血漿纖維蛋白原等血液學指標以及血清丙氨酸氨基轉移酶、天冬氨酸氨基轉移酶、堿性磷酸酶、乳酸脫氫酶、總膽紅素、尿素氮、肌酐、總蛋白、白蛋白、血糖、總膽固醇、甘油三脂、肌酸磷酸激酶，血清鈣、磷、血清鉀、鈉和氯等血液生化指標。在給藥第14天和第21天分別對每組各1/2的動物剖檢，進行組織病理學檢查及骨髓細胞計數分類。結果：（1）給藥組大鼠在給藥后出現(xiàn)呼吸抑制、震顫等毒性反應，高劑量組較為嚴重，但均在20-30分鐘內恢復正常。（2）連續(xù)灌胃KCN14天對大鼠的體重沒有明顯影響。（3）給藥后各組動物的進食量沒有明顯變化。（4）KCN對大鼠的血液學指標沒有明顯影響。（5）KCN對大鼠的血液生化指標無明顯影響。（6）KCN給藥后，對大鼠的骨髓細胞沒有明顯影響。（7）停藥期時，高劑量組雌性大鼠腦組織的絕對重量及相對重量均顯著下降；部分高劑量組大鼠大腦皮質層小膠質細胞灶性增生，形成膠質小結樣病變；灰白質交界處個別神經元細胞變性壞死，神經膠質細胞灶性浸潤填充；更有個別動物大腦白質呈灶狀空泡樣變性，伴隨有髓纖維數量的減少；部分動物甚至出現(xiàn)大腦基底部白質的灶性脫髓鞘樣改變；少量動物海馬回神經細胞銳減，甚至出現(xiàn)局灶性海馬神經細胞大量脫落，且脫落區(qū)域可見大量神經膠質細胞浸潤填充。其余臟器未見明顯異常�；謴推跁r未見明顯異常。結論：對大鼠連續(xù)14天的灌胃KCN試驗，，給藥組大鼠在給藥時出現(xiàn)嚴重的毒性反應，毒性靶器官為中樞神經系統(tǒng)，毒性作用可逆。 第二部分KCN神經毒性機制研究 目的：結合KCN14天的灌胃試驗，對Wistar大鼠大腦組織進行全基因組變化研究，找到KCN神經毒性反應可能涉及的信號通路；并對大鼠的血液和尿液進行代謝組學分析，揭示KCN給藥對代謝產物的影響。方法：（1）基因組學研究：取12只大鼠（停藥期0mg/kg劑量組4只，停藥期10mg/kg劑量組4只，恢復期10mg/kg劑量組4只）的大腦組織，提取RNA，標記并擴增后與基因表達譜芯片進行雜交，經掃描分析篩選出給藥組與對照組表達差異的基因。（2）代謝組學分析：收集對照組、低劑量組和高劑量組大鼠(每組8只)給藥第14天的尿液及血清樣本，做代謝組學分析。篩選出能體現(xiàn)KCN毒性的生物標志物。結果：（1）芯片雜交信號清晰，背景低，信噪比高，基因芯片檢測和聚類分析結果有效。（2）經過T-test檢驗，篩選出顯著上調（信號比值≥2）或顯著下調（信號比值"f0.5）的差異表達基因289個。（3）按照基因功能分類體系（Gene Ontology, GO）分析差異表達基因，結果顯示，差異基因體現(xiàn)的生物學功能多樣，包括正向調節(jié)成神經細胞的增殖、大腦皮層發(fā)育、負向調節(jié)神經細胞凋亡、正向調節(jié)細胞增殖、鈣離子應答過程、動作電位的調節(jié)、細胞分化的調節(jié)等。（4）導致KCN神經毒性的可能信號通路有：粘著斑信號通路、多巴胺能突觸信號通路、谷氨酸能突觸信號通路、細胞外基質受體相互作用信號通路、神經受體配體相互作用信號通路、神經營養(yǎng)因子信號通路、鈣信號通路。（5）代謝組學分析：大鼠血清中磷酸膽堿，甘油磷酸膽堿，谷氨酰胺及甲基組氨酸的水平顯著升高，賴氨酸的水平顯著降低；大鼠尿液中氧化三甲胺，對羥基乙酸苯酯，羥丙茶堿，馬尿酸鹽，二甲胺水平相對溶媒對照組顯著增加，丁二酸，乳酸鹽，肌酸，瓜氨酸，乙酸鹽水平顯著降低。結論：（1）KCN的中樞神經毒性作用可能和其以下特性有關：KCN神經毒性的產生與神經細胞內鈣穩(wěn)態(tài)失調，Ca2+內流有關；KCN對粘著斑信號通路和細胞外基質受體相互作用信號通路的ITGB6有一定的調控作用，影響細胞與細胞間的相互作用；KCN對多巴胺能突觸和谷氨酸能突觸信號通路中GRIN2A有一定的調控作用，影響神經細胞間的突觸傳遞和信號轉導；對神經營養(yǎng)因子信號通路中NTRK2基因有一定的調控作用，影響神經營養(yǎng)因子與酪氨酸激酶受體Trk的結合，進而影響神經細胞的功能。（2）KCN神經毒性的產生，推測是有多條信號通路異常引起。其中最重要的機制可能是由于鈣穩(wěn)態(tài)失調，引發(fā)Ca2+內流，導致生物膜脂質過氧化，影響神經細胞的突觸傳遞和信號轉導，產生CNS毒性。（3）大鼠代謝組學研究結果提示，血清谷氨酰胺、賴氨酸及尿液中檸檬酸鹽、丁二酸、乙酸鹽水平變化可以作為KCN神經毒性的生物標志物，但需要進一步的研究證實。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114

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