CYP2E1在二甲基甲酰胺致小鼠脏急性损伤中的作用及相关机制

发布时间：2018-06-18 00:07

本文选题：二甲基甲酰胺 + Cyp2e1基因敲除小鼠　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：目的二甲基甲酰胺(DMF)是一种具有很强溶解能力的有机物质,曾被誉为“万能溶剂”。在室温条件下,DMF能够与水、酮、酯、醚、醇、氯化烃和芳烃等有机物以任何比例完全混溶。在工业中,可以作为聚胺酯人造革及合成革的加工溶剂,以及作为萃取剂参与医药和染料的生产过程,用途十分广泛;DMF有一定的挥发性,在工业企业的生产过程中,如果没有合理的防护,DMF会通过呼吸及皮肤接触进入机体,对职业工人的消化系统、生殖系统、免疫系统等造成健康危害。DMF进入机体后,肝脏为其靶器官,能够导致肝脏出现ALT和AST等肝酶升高、肝脏系数增大、肝细胞坏死等肝脏损伤表现,并能特异性地产生血红蛋白加合物(NMHb)。在DMF的代谢过程中,CYP450家族的Cyp2e1酶起到了非常重要的作用。本研究通过敲除小鼠Cyp2e1基因对CYP2E1在DMF代谢中的作用进行研究。通过建立DMF致小鼠急性肝毒性效应模型,探讨DMF所致的急性肝毒性效应及CYP2E1在代谢过程中的作用。并通过检测氧化抗氧化和内质网应激相关指标的水平,探究产生肝损伤的可能机制。方法1、建立Cyp2e1基因敲除小鼠:通过TALEN技术对小鼠Cyp2e1基因进行敲除,显微注射导入到受体C57BL/6母鼠中,产生基因敲除杂合子。通过杂合子之间的交配,产生Cyp2e1基因敲除纯合型小鼠。通过一代测序鉴定小鼠基因型、Western blot法检测Cyp2e1蛋蛋表达含量、高效液相色谱法检测Cyp2e1酶活性;2、建立DMF致小鼠急性肝毒性效应模型:选取12周龄基因敲除纯合型雌雄性小鼠各32只,及相等数量相应周龄的C57BL/6野生型小鼠。将小鼠按照基因型和性别不通过分成雄性野生型、雌性野生型、雄性纯合型、雌性纯合型4个大组,每大组中的小鼠随机分为对照组、染毒后24 h组、染毒后48 h组和染毒后72 h组。染毒剂量为1500mg/kg·bw,单次灌胃染毒后,根据不同恢复时间处死动物;并通过计算肝脏系数、检测肝酶水平和观察肝脏病理组织学推断DMF所致肝脏急性毒性效应;3、血红蛋白加合物和CYP2E1:通过高效液相色谱法检测血液中NMHb的含量观察DMF在小鼠体内的代谢情况;通过检测Cyp2e1mRNA水平、Cyp2e1蛋白水平和酶活性提示CYP2E1在DMF代谢中的作用;4、小鼠氧化抗氧化说和内质网应激的相关指标:选取GSH和MDA作为氧化应激的指标,选取SOD和GSH-Px作为抗氧化酶指标;选取GRP94和GRP78作为内质网应激指标。结果1、基因敲除小鼠的建立:一代测序DNA序列图谱可见Cyp2e1基因敲除纯合型小鼠序列第166位插入了一个碱基C且无杂峰;Western blot检测纯合型小鼠Cyp2el蛋白表达量远低于野生型小鼠;酶活性检测可见纯合型小鼠与酶自身性质相关的Km值与野生型相差不大,与酶含量相关的Vmax远低于野生型小鼠;2、DMF致小鼠急性肝毒性效应:雌雄性野生型小鼠在染毒后三个时间点出现肝脏系数、肝酶ALT和AST水平显著升高,纯合型小鼠肝酶水平改变无统计学差异。野生型小鼠肝脏病理在染毒后出现肝细胞水样变、大片性细胞坏死伴炎细胞浸润,纯合型小鼠在病变的范围和程度上均较野生型小鼠轻;3、血红蛋白加合物和CYP2E1:NMHb检出有特异性,对照组不能检出;雄性野生型小鼠暴露DMF后24 h已基本完成DMF代谢,生成NMHb;雌性野生型小鼠染毒后24 h时NMHb生成量较少,NMHb水平在48 h急剧升高,提示DMF代谢重要发生在24-48 h;纯合型雌雄性小鼠均有少量NMHb生成。PCR检测小鼠Cyp2e1mRNA水平可见染毒后有mRNA水平变化,Western blot实验未见纯合型组明显Cyp2el蛋白条带,野生型小鼠Cyp2e1蛋白在染毒后有先升高后降低的趋势,提示DMF可能引起CYP2E1转录和翻译水平的调控,并可能出现CYP2E1自身抑制;雌雄性野生型小鼠在染毒后48h出现Cyp2e1酶活性下降,提示可能与自身抑制有关;雄性纯合型小鼠酶活性本底值较低,染毒后有升高趋势;4、DMF所致氧化应激和内质网应激:DMF暴露会导致肝脏内GSH含量降低,MDA含量升高,激活体内SOD和GSH-Px等抗氧化酶的活性。内质网应激相关蛋白GRP94和GRP78等蛋白水平在DMF暴露后升高;5、性别差异:血生化结果中雌性野生型小鼠在DMF染毒后出现甘油三酯和胆固醇的升高,而在雄性野生型小鼠中未见两项指标的改变。野生型小鼠因坏死明显而肝脏病理组织学检测未见性别差异,但在纯合型小鼠中差异明显,表现为雄性纯合型小鼠病理组织学改变以肝细胞肿胀为主,雌性纯合型小鼠病理组织学改变以肝脏脂肪变性为主。NMHb观察到DMF的代谢情况,雄性野生型小鼠的DMF代谢主要发生在0-24 h,雌性野生型小鼠DMF代谢主要发生在24-48 h;结论1、成功构建Cyp2e1基因小鼠纯合型;2、CYP2E1在DMF致小鼠急性肝毒性效应中起到了毒物的代谢活化、产生活性氧等重要作用;3、内质网应激为可能的DMF致小鼠肝脏损伤的机制。
[Abstract]:Objective two methyl formamide (DMF) is a kind of organic substance with strong solubility. It was once known as "universal solvent". At room temperature, DMF can be completely mixed with water, ketone, ester, ether, alcohol, chlorinated hydrocarbon and aromatics in any proportion. In industry, it can be used as a processing solvent for polyamines and synthetic leather. The use of extractants in the production of pharmaceuticals and dyes is very extensive; DMF has a certain volatility. In the production process of industrial enterprises, if there is no reasonable protection, DMF will enter the body through breathing and skin contact, causing health hazards to occupational workers, such as digestive system, reproductive system, immune system and so on, when.DMF enters the body. The liver is its target organ, which can lead to liver enzymes such as ALT and AST increase, liver coefficient increased, liver necrosis and other liver damage and specific real estate hemoglobin adducts (NMHb). In the metabolic process of DMF, the Cyp2e1 enzyme in the CYP450 family has played a very important role. This study knocks the Cyp2e1 gene in mice by knocking off the Cyp2e1 gene. The role of CYP2E1 in DMF metabolism was studied. The acute hepatotoxicity effect model induced by DMF in mice was established to explore the acute hepatotoxicity effect of DMF and the role of CYP2E1 in the metabolic process. The possible mechanism of producing liver injury was explored by detecting the level of oxidation oxidation resistance and endoplasmic reticulum stress. Method 1, establish Cyp2 E1 gene knockout mice: the mouse Cyp2e1 gene was knocked out by TALEN technique, and the gene knockout heterozygote was produced by microinjection into the recipient C57BL/6 mouse. Through mating among the heterozygotes, the Cyp2e1 gene was knocked out of the homozygous mice. The gene type of the mouse was identified by a generation sequence and the Western blot method was used to detect the expression of the Cyp2e1 eggs. The activity of Cyp2e1 enzyme was detected by HPLC; 2, the model of acute hepatotoxicity induced by DMF was established: 32 mice of 12 weeks old gene knockout homozygous male and female mice were selected, and the equivalent number of C57BL/6 wild type mice corresponding to the corresponding weeks age. The mice were divided into male wild type, female wild type and male homozygous in accordance with genotype and sex. The mice in each group were divided into 4 groups. The mice in each group were randomly divided into the control group, 24 h groups after exposure, 48 h groups after exposure and 72 h after exposure. The dose was 1500mg/kg. BW, and the animals were killed according to the different recovery time. The liver enzyme level was detected and the liver histopathology was observed to infer DMF by calculating the liver coefficient. Acute toxic effects of liver were induced; 3, hemoglobin adducts and CYP2E1: measured the NMHb content in blood by high performance liquid chromatography to observe the metabolism of DMF in mice; by detecting Cyp2e1mRNA level, Cyp2e1 protein level and enzyme activity suggested the role of CYP2E1 in DMF metabolism; and 4, oxidative antioxidant said and endoplasmic reticulum stress in mice. GSH and MDA were selected as indicators of oxidative stress, SOD and GSH-Px were selected as indicators of antioxidant enzymes, and GRP94 and GRP78 were selected as endoplasmic reticulum stress indicators. Results 1, gene knockout mice were established: one generation sequencing DNA sequence showed that 166th bits of Cyp2e1 gene knockout homozygous mice were inserted into a base C and no heterozygosity. Western blot showed that the expression of Cyp2el protein in homozygous mice was far lower than that of wild type mice; the enzyme activity detection showed that the Km value related to the enzyme itself was not much different from the wild type, and the Vmax related to the enzyme content was far lower than that of the wild type mice; 2, DMF induced acute hepatotoxicity effect in mice: the male and female wild type mice were infected. Liver coefficient, liver enzyme ALT and AST level increased significantly at three time points, and there was no significant difference in liver enzyme level in homozygous mice. Liver pathology in the wild type mice showed liver cell water change, large cell necrosis and inflammatory cells infiltration, and the range and degree of homozygous mice were lighter than those of wild type mice; 3, blood. The detection of erythroprotein adducts and CYP2E1:NMHb was specific, and the control group could not be detected. After exposure to DMF in male wild type mice, 24 h had basically completed DMF metabolism and produced NMHb. The female wild type mice had less NMHb generation and NMHb level at 48 h after 24 h, suggesting that DMF metabolism was important in 24-48 h; homozygous male and female mice were all There was a small amount of NMHb generated by.PCR to detect Cyp2e1mRNA level in mice, and there was a change in the level of mRNA. The Western blot experiment did not see the obvious Cyp2el protein band in the homozygous group. The Cyp2e1 protein in the wild type mice increased and then decreased after exposure, suggesting that DMF might lead to the regulation of the CYP2E1 transcripts and the level of translation, and may appear CYP2E1. Body inhibition; the Cyp2e1 enzyme activity of 48h in male and female wild type mice decreased, suggesting that it may be related to the inhibition of self inhibition; the bottom value of enzyme activity in male homozygous mice was lower, and the tendency to increase after exposure; 4, DMF induced oxidative stress and endoplasmic reticulum stress: DMF exposure could lead to the decrease of GSH content in the liver, the increase of MDA content and activation of SOD in the body. The activity of antioxidant enzymes, such as GSH-Px, and the levels of endoplasmic reticulum stress related protein GRP94 and GRP78 increased after DMF exposure; 5, sex difference: the increase of triglyceride and cholesterol in the female wild type mice after DMF was found in the blood biochemical results, but no change in the two indexes in the male wild type mice. There was no gender difference in the liver histopathology test, but in the homozygous mice, the male homozygous mice were characterized by histopathological changes and liver cell swelling, and the pathological changes of the female homozygous mice were based on the fatty degeneration of the liver.NMHb to observe the metabolism of DMF, and the DM of the male wild type mice. The metabolism of F mainly occurred in 0-24 h, and the metabolism of DMF in female wild type mice mainly occurred at 24-48 h; conclusion 1, the Cyp2e1 Gene Mouse homozygous type was successfully constructed; 2, CYP2E1 played an important role in the metabolic activation of toxic substances in the acute hepatotoxicity induced by DMF in mice and the production of active oxygen, and 3, endoplasmic reticulum stress was a possible DMF causing liver injury in mice. System.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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