苯中毒的代谢特征与造血毒作用机制的研究

发布时间：2018-06-12 15:07

本文选题：苯中毒 + 造血毒性　；参考：《东南大学》2016年博士论文

【摘要】：苯暴露可引起造血系统恶性肿瘤,苯造血毒性的分子机制迄今尚未完全阐明。研究苯暴露早期阶段到苯造血毒性发生发展过程中的关键事件及相关分子机制可以为苯中毒的诊断干预及早期预防提供科学依据。系统生物学研究显示,机体细胞在生理病理不同状态及疾病发生发展不同阶段都具有特异性的代谢特征。本研究拟通过建立苯中毒小鼠模型,获得小鼠外周循环及造血细胞的代谢特征,结合毒理学实验和生物信息学结果分析特征性改变内源性小分子物质的相关代谢通路;进一步研究苯造血毒性相关的特异性代谢通路变化及作用机制;在此基础上探讨乙酰左旋肉碱干预是否影响苯暴露所致造血毒性、线粒体功能障碍和氧化应激;最后初步探讨了苯暴露对癌基因MDS-Evi1表达的影响以及该基因在造血干细胞凋亡调控中的作用机制。第一章苯中毒小鼠模型的建立及造血抑制毒性研究使用C3H/He小鼠,苯暴露剂量为0、150及300 mg/kg·bw,皮下注射方式染毒,每天1次,连续5天,共染毒4周,构建苯中毒小鼠模型,模型构建后进行小鼠外周血常规、骨组织病理学、骨髓涂片、造血干细胞比例及造血集落形成能力检查。结果显示,通过皮下注射的染毒方式可成功构建苯中毒小鼠模型,苯暴露对小鼠神经系统初期为兴奋效应,后期为抑制作用;苯暴露降低小鼠外周血白细胞、红细胞、血红蛋白和血小板计数,引起贫血,组织病理学检查结果显示苯引起小鼠骨髓和脾脏造血抑制,骨髓涂片可见苯组小鼠骨髓细胞形态及增生异常、原始细胞比例明显增加;此外,苯暴露还可引起小鼠LSK细胞比例下降,造血干祖细胞向GEMM和GM分化能力降低。以上结果表明苯暴露可引起小鼠外周血细胞计数降低、骨髓和脾脏造血抑制,骨髓细胞形态和增生分化异常;引起LSK细胞比例下降,GEMM和GM造血集落形成能力降低。第二章基于LC-MS研究苯暴露小鼠的代谢特征使用LC/MS技术,研究苯暴露小鼠体循环(尿液、血浆)及靶器官(骨髓细胞)代谢特征,比较特异性内源性小分子代谢产物及其涉及的代谢通路;结合血液毒性评价及代谢特征,筛选潜在的尿液和血浆中标志血液系统早期损伤的代谢标志物以及与苯造血毒作用相关的特异性代谢通路;此外,比较获得在动物模型骨髓细胞和血浆样品中发现的共同差异代谢产物,在苯暴露人群血浆样本中进行验证。成功建立基于LC-MS的小鼠尿液、血浆和骨髓细胞代谢组学分析平台,结果显示苯暴露扰乱尿液嘌呤、脂肪酸、色氨酸、谷酰基苯丙氨酸和亚精胺代谢通路,干扰血浆色氨酸、组氨酸代谢、γ-谷氨酰循环、脂肪酸氧化以及脂肪酸代谢通路,导致骨髓细胞酪氨酸和苯丙氨酸代谢、赖氨酸分解、肉碱合成以及脂肪酸氧化代谢通路紊乱;比较苯暴露小鼠血浆和骨髓细胞中的差异性代谢通路,发现脂肪酸β氧化通路中的代谢分子均检测到一致的改变,在收集的正常人群、苯暴露对照人群及苯暴露血象异常人群血浆样本中进行验证,发现苯暴露组人群血浆中左旋肉碱含量显著下降,而乙酰左旋肉碱未发生明显变化。以上结果提示苯暴露可干扰小鼠尿液、血浆和骨髓细胞中不同的代谢通路,其中,脂肪酸β氧化代谢通路是在外周样本血浆及靶器官骨髓中的共同差异代谢通路,可能与苯造血毒性效应密切相关。第三章苯暴露对脂肪酸氧化代谢通路的影响及机制研究将C3H/He小鼠暴露于0、20、40、80、160mg/kg·bw苯,以皮下注射方式染毒,每天1次,连续5天,共染毒4周,检测苯暴露对小鼠骨髓细胞脂肪酸代谢通路关键酶mRNA水平和蛋白水平的影响,同时检测对线粒体功能和氧化应激的影响。结果显示,苯暴露可影响脂肪酸氧化通路中肉碱穿梭酶和β氧化关键酶mRNA表达,蛋白水平的验证表明苯暴露可增强小鼠骨髓细胞肉碱穿梭蛋白Cpt1a和Crat的表达,使脂肪酸β氧化通路中Acaa2、A1dh112、Acadv1、Crot、Echs1和Hadha蛋白表达增加;苯暴露引起小鼠骨髓细胞中左旋肉碱、ATP和线粒体膜电位水平降低,同时还可引起H2O2、MDA和ROS等氧化损伤指标水平增高。以上结果提示苯暴露可影响脂肪酸氧化通路中肉碱穿梭及β氧化过程,引起线粒体功能障碍和氧化应激。第四章乙酰左旋肉碱干预对苯所致造血毒性的影响使用100 mg/kg·bw和200 mg/kg·bw左旋乙酰肉碱干预150 mg/kg·bw苯暴露小鼠,观察乙酰左旋肉碱对苯所致造血毒性、线粒体功能障碍和氧化应激的影响。结果显示,左旋乙酰肉碱对苯暴露导致的小鼠外周血细胞减少无明显作用,但是200 mg/kg·bw左旋乙酰肉碱干预可以明显增加苯暴露组小鼠全骨髓细胞数量、LSK造血干细胞比例和Lini-C-kit-Sca-1+造血祖细胞比例;乙酰左旋肉碱干预可以在一定程度上减轻苯所致的线粒体损伤,可降低苯暴露所致H202、MDA和ROS增加;200 mg/kg乙酰左旋肉碱干预可明显降低苯暴露小鼠骨髓细胞尾部DNA含量、尾距和尾长,减轻DNA损伤。以上结果提示乙酰左旋肉碱干预可在一定程度上减轻苯所致小鼠全骨髓细胞、LSK造血干细胞损伤,其作用机制可能涉及减少苯暴露所致氧化应激,降低苯诱导DNA损伤。第五章MDS-Evi1基因与苯毒性关系初探及其参与骨髓造血干细胞凋亡调控的研究使用0、1μm、5μm和10μm1,4-苯醌染毒小鼠骨髓lin-细胞24小时,检测细胞增殖、细胞凋亡以及MECOM基因表达,同时在苯暴露小鼠骨髓细胞中检测MECOM蛋白的表达;随后建立在ME表达细胞中特异性敲除Bax和Bak基因的MEm2小鼠,观察LSK细胞数量、细胞增殖、细胞凋亡、集落形成能力以及竞争性骨髓移植后LT-HSCs的重建能力。结果显示,5μm和1Oμm1,4-苯醌组细胞存活率明显下降,1μM 1,4-苯醌组细胞的凋亡率最大;随着染毒剂量的增加,细胞MECOM基因表达增加;体内实验结果显示160 mg/kg·bw苯暴露引起MECOM蛋白表达显著增加。ME基因敲除小鼠LSK细胞的数量下降,增殖加速,凋亡率增加,且这种凋亡是造血干细胞自发性的;在ME表达细胞中特异性地敲除Bax和Bak基因可使LSK细胞数量和增殖正常,骨髓细胞CFU-GEMM和CFU-GM集落形成能力增加,可以挽救MEm2/m2小鼠LT-HSCs重建能力。以上结果提示苯暴露可增加癌基因MDS-Evi1表达水平,该基因通过Bax和Bak通路参与造血干细胞增殖、凋亡和重建功能调控。总结综上,本研究成功建立苯中毒小鼠模型,结果表明苯暴露抑制造血,同时可扰乱小鼠尿液、血浆和骨髓细胞不同代谢通路,其中,脂肪酸β氧化代谢通路可能与苯造血毒性密切相关;进一步研究表明苯暴露引起脂肪酸氧化通路中肉碱穿梭及β氧化过程中关键酶表达增加、线粒体功能障碍和氧化应激;而给予乙酰左旋肉碱干预可在一定程度上减轻苯所致小鼠造血系统损伤,其作用机制可能涉及减少苯暴露所致氧化应激和DNA损伤;此外,苯暴露可增加癌基因MDS-Evi1表达水平,该基因通过Bax和Bak通路参与造血干细胞增殖、凋亡和重建功能调控。
[Abstract]:Benzene exposure can cause malignant tumor of hematopoietic system. The molecular mechanism of hematopoietic toxicity of benzene has not been fully elucidated so far. The key events and related molecular mechanisms in the study of the early stage of benzene exposure to the development of benzene hematopoiesis and related molecular mechanisms can provide scientific basis for the diagnosis and early prevention of benzene poisoning. Somatic cells have specific metabolic characteristics in different stages of physiological and pathological conditions and different stages of disease and development. This study aims to establish a mouse model of benzene poisoning to obtain the metabolic characteristics of peripheral circulation and hematopoietic cells in mice, and combine the toxicological experiments and bioinformatics results to analyze the characteristics of endogenous small molecular substances. Related metabolic pathways; further study on the specific metabolic pathways related to the hematopoietic toxicity of benzene and the mechanism of action; and on the basis of this, whether acetyl L-carnitine intervention affects hematopoiesis, mitochondrial dysfunction and oxidative stress induced by benzene exposure; finally, the effect of benzene exposure on the expression of oncogene MDS-Evi1 and the effect of benzene exposure on the expression of oncogene are discussed. The mechanism of gene in the regulation of hematopoietic stem cell apoptosis. In Chapter 1, the establishment of the mice model of benzene poisoning and the study of hematopoietic inhibition toxicity were used in C3H/He mice, the exposure dose of benzene was 0150 and 300 mg/kg BW, the subcutaneous injection was poisoned, 1 times a day for 5 days and a total of 4 weeks were infected, and the model of benzene poisoning mice was constructed and the model of mice was constructed outside the mice. Peripheral blood routine, bone histopathology, bone marrow smear, hematopoietic stem cell ratio and hematopoietic colony forming ability examination. The results showed that the mice model of benzene poisoning could be successfully constructed by subcutaneous injection. Benzene exposure was excited in the early stage of the nervous system and later was inhibited, and benzene exposure decreased peripheral blood leukocytes in mice. Erythrocyte, hemoglobin and platelet count, causing anemia. Histopathological examination results showed that benzene caused bone marrow and spleen hematopoiesis in mice. Bone marrow smears showed abnormal morphology and proliferation of bone marrow cells in benzene group, and the proportion of primitive cells increased significantly. In addition, benzene exposure could cause the decrease of LSK cells in mice and hematopoietic progenitor cells. The differentiation of GEMM and GM decreased. The above results showed that benzene exposure could reduce the number of peripheral blood cells in mice, bone marrow and spleen hematopoiesis, bone marrow cell morphology and proliferation and differentiation, the decrease of LSK cells and the decrease of GEMM and GM hematopoiesis. The second chapter was based on LC-MS to study the metabolic characteristics of mice exposed to benzene. LC/MS technology studies the metabolic characteristics of benzene exposed mice body circulation (urine, plasma) and target organs (bone marrow cells), compares the specific endogenous small molecular metabolites and their metabolic pathways, and combines the evaluation of blood toxicity and metabolic characteristics to screen the metabolic markers for the early damage of the blood system in the urine and blood plasma. Specific metabolic pathways related to the effect of benzene on hematopoiesis; in addition, the common differential metabolites found in the animal model bone marrow cells and plasma samples were compared in the plasma samples of the exposed population of benzene. A successful establishment of a LC-MS based mouse urine, plasma and bone marrow cell metabolic analysis platform was established. The results showed that benzene exposure was exposed. Disrupting urine purine, fatty acids, tryptophan, glutamyl phenylalanine and arginine metabolic pathways, interfering with plasma tryptophan, histidine metabolism, gamma glutamyl cycle, fatty acid oxidation and fatty acid metabolism pathway, leading to tyrosine and phenylalanine metabolism in bone marrow cells, lysine decomposition, carnitine synthesis, and fatty acid oxidation metabolic pathways turbulence The metabolic pathways in the plasma and bone marrow cells of mice exposed to benzene were compared, and the metabolic molecules in the fatty acid beta oxidation pathway were detected by the same changes. In the normal population, the benzene exposed control population and the blood samples from the abnormal group of benzene exposed hemogram were tested, and the plasma L-carnitine in the exposed group of benzene was found. The content of acetyl L-carnitine did not change significantly. The above results suggested that benzene exposure could interfere with the different metabolic pathways in the urine, plasma and bone marrow cells of mice, in which the fatty acid beta oxidation pathway is a common metabolic pathway in the peripheral blood samples and the target organ bone marrow, and may be closely related to the toxic effect of benzene on hematopoiesis. The influence and mechanism of benzene exposure on the oxidative metabolic pathway of fatty acids in third chapters, C3H/He mice were exposed to 0,20,40,80160mg/kg BW benzene, exposed to subcutaneous injection, 1 times a day for 5 days, for 4 weeks, and the effects of benzene exposure on the level of mRNA and protein level of the key enzymes of fatty acid metabolite pathway in mice bone marrow cells were detected. The results showed that benzene exposure could affect the expression of carnitine shuttle and beta oxidation key enzyme mRNA in the fatty acid oxidation pathway, and the protein level showed that benzene exposure could enhance the expression of carnitine shuttle protein Cpt1a and Crat in mouse bone marrow cells and make Acaa2, A1dh1 in the fatty acid beta oxidation pathway. 12, Acadv1, Crot, Echs1 and Hadha protein expression increased; benzene exposure caused the decrease of L-carnitine, ATP and mitochondrial membrane potential in mouse bone marrow cells, and also increased the levels of H2O2, MDA, ROS and other oxidative damage indicators. The above results suggest that benzene exposure can affect the process of carnitine shuttle and beta oxidation in the fatty acid oxidation pathway, causing lines. The effect of acetyl L-carnitine intervention on hematopoiesis induced toxicity of benzene in fourth chapters: the effects of 100 mg/kg. BW and 200 mg/kg. BW L-acetylcarnitine on 150 mg/kg. BW benzene exposure mice. The effects of acetyl L-carnitine on hematopoiesis, mitochondrial dysfunction and oxidative stress were observed. Acetyl carnitine had no significant effect on the decrease of peripheral blood cells in mice caused by benzene exposure, but the intervention of 200 mg/kg. BW levoacetyl carnitine could significantly increase the number of all bone marrow cells in the mice exposed to benzene, the proportion of LSK hematopoietic stem cells and the proportion of Lini-C-kit-Sca-1+ hematopoietic progenitor cells, and the ethyl L-carnitine intervention could be reduced to a certain extent. The mitochondrial damage caused by light benzene could reduce the increase of H202, MDA and ROS caused by benzene exposure, and the intervention of 200 mg/kg acetyl L-carnitine could significantly reduce the DNA content, tail length and tail length of mice bone marrow cells exposed to benzene, and reduce DNA damage. The results suggested that acetyl L-carnitine intervention could reduce the total bone marrow cells of benzene induced mice to a certain extent, LSK hematopoietic stem cell injury, its mechanism may be involved in reducing oxidative stress caused by benzene exposure and reducing benzene induced DNA damage. Fifth the relationship between MDS-Evi1 gene and benzene toxicity and its involvement in the regulation of bone marrow hematopoietic stem cell apoptosis using 0,1 mu m, 5 m and 10 micron benzquinone in mice bone marrow lin- cells for 24 hours, and detection of cell proliferation. Colonization, apoptosis and expression of MECOM gene, and the expression of MECOM protein in bone marrow cells exposed to benzene, and subsequently established in MEm2 mice that specifically knock out Bax and Bak genes in ME expression cells, observe the number of LSK cells, cell proliferation, cell apoptosis, colony forming ability and LT-HSCs reconstruction after competitive bone marrow transplantation. The results showed that the cell survival rate of 5 m and 1O m1,4- quinone group decreased significantly, the apoptosis rate of the 1 M 1,4- benzone group was the largest, and the expression of MECOM gene in the cells increased with the increase of dose. The experimental results showed that the expression of MECOM protein in the 160 mg/kg / BW benzene exposure significantly increased the number of LSK cells in the.ME gene knockout mice. The proliferation accelerated and the apoptosis rate increased, and the apoptosis was spontaneous. The specific knockout of the Bax and Bak genes in the ME expression cells could make the number and proliferation of LSK cells normal, the CFU-GEMM and CFU-GM colony formation ability of the bone marrow cells increased, which could save the LT-HSCs reconstruction ability of MEm2/m2 mice. The above results suggest that the benzene exposure can be increased. The expression level of oncogene MDS-Evi1, which participates in the proliferation, apoptosis and reconstruction function of hematopoietic stem cells through Bax and Bak pathway, has been successfully established in this study. The results show that benzene exposure inhibits hematopoiesis and can disturb the different metabolic pathways of urine, plasma and bone marrow cells in mice, including fatty acid beta oxygen. The metabolic pathway may be closely related to the hematopoiesis of benzene. Further studies have shown that the expression of key enzymes in the process of carnitine shuttle and beta oxidation in the fatty acid oxidation pathway is increased, and the mitochondrial dysfunction and oxidative stress are increased in the fatty acid oxidation pathway, while acetyl L-carnitine intervention can reduce the hematopoietic system damage caused by benzene in mice to a certain extent. Its mechanism may be involved in reducing oxidative stress and DNA damage caused by benzene exposure. In addition, benzene exposure can increase the expression of oncogene MDS-Evi1, which is involved in the regulation of hematopoietic stem cell proliferation, apoptosis and reconstruction through the Bax and Bak pathways.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R135.12

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