本文選題：二甲基甲酰胺 + Cyp2e1基因敲除小鼠　； 參考：《中國(guó)疾病預(yù)防控制中心》2017年碩士論文

【摘要】：目的二甲基甲酰胺(DMF)是一種具有很強(qiáng)溶解能力的有機(jī)物質(zhì),曾被譽(yù)為“萬(wàn)能溶劑”。在室溫條件下,DMF能夠與水、酮、酯、醚、醇、氯化烴和芳烴等有機(jī)物以任何比例完全混溶。在工業(yè)中,可以作為聚胺酯人造革及合成革的加工溶劑,以及作為萃取劑參與醫(yī)藥和染料的生產(chǎn)過(guò)程,用途十分廣泛;DMF有一定的揮發(fā)性,在工業(yè)企業(yè)的生產(chǎn)過(guò)程中,如果沒(méi)有合理的防護(hù),DMF會(huì)通過(guò)呼吸及皮膚接觸進(jìn)入機(jī)體,對(duì)職業(yè)工人的消化系統(tǒng)、生殖系統(tǒng)、免疫系統(tǒng)等造成健康危害。DMF進(jìn)入機(jī)體后,肝臟為其靶器官,能夠?qū)е赂闻K出現(xiàn)ALT和AST等肝酶升高、肝臟系數(shù)增大、肝細(xì)胞壞死等肝臟損傷表現(xiàn),并能特異性地產(chǎn)生血紅蛋白加合物(NMHb)。在DMF的代謝過(guò)程中,CYP450家族的Cyp2e1酶起到了非常重要的作用。本研究通過(guò)敲除小鼠Cyp2e1基因?qū)YP2E1在DMF代謝中的作用進(jìn)行研究。通過(guò)建立DMF致小鼠急性肝毒性效應(yīng)模型,探討DMF所致的急性肝毒性效應(yīng)及CYP2E1在代謝過(guò)程中的作用。并通過(guò)檢測(cè)氧化抗氧化和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)指標(biāo)的水平,探究產(chǎn)生肝損傷的可能機(jī)制。方法1、建立Cyp2e1基因敲除小鼠:通過(guò)TALEN技術(shù)對(duì)小鼠Cyp2e1基因進(jìn)行敲除,顯微注射導(dǎo)入到受體C57BL/6母鼠中,產(chǎn)生基因敲除雜合子。通過(guò)雜合子之間的交配,產(chǎn)生Cyp2e1基因敲除純合型小鼠。通過(guò)一代測(cè)序鑒定小鼠基因型、Western blot法檢測(cè)Cyp2e1蛋蛋表達(dá)含量、高效液相色譜法檢測(cè)Cyp2e1酶活性;2、建立DMF致小鼠急性肝毒性效應(yīng)模型:選取12周齡基因敲除純合型雌雄性小鼠各32只,及相等數(shù)量相應(yīng)周齡的C57BL/6野生型小鼠。將小鼠按照基因型和性別不通過(guò)分成雄性野生型、雌性野生型、雄性純合型、雌性純合型4個(gè)大組,每大組中的小鼠隨機(jī)分為對(duì)照組、染毒后24 h組、染毒后48 h組和染毒后72 h組。染毒劑量為1500mg/kg·bw,單次灌胃染毒后,根據(jù)不同恢復(fù)時(shí)間處死動(dòng)物;并通過(guò)計(jì)算肝臟系數(shù)、檢測(cè)肝酶水平和觀察肝臟病理組織學(xué)推斷DMF所致肝臟急性毒性效應(yīng);3、血紅蛋白加合物和CYP2E1:通過(guò)高效液相色譜法檢測(cè)血液中NMHb的含量觀察DMF在小鼠體內(nèi)的代謝情況;通過(guò)檢測(cè)Cyp2e1mRNA水平、Cyp2e1蛋白水平和酶活性提示CYP2E1在DMF代謝中的作用;4、小鼠氧化抗氧化說(shuō)和內(nèi)質(zhì)網(wǎng)應(yīng)激的相關(guān)指標(biāo):選取GSH和MDA作為氧化應(yīng)激的指標(biāo),選取SOD和GSH-Px作為抗氧化酶指標(biāo);選取GRP94和GRP78作為內(nèi)質(zhì)網(wǎng)應(yīng)激指標(biāo)。結(jié)果1、基因敲除小鼠的建立:一代測(cè)序DNA序列圖譜可見(jiàn)Cyp2e1基因敲除純合型小鼠序列第166位插入了一個(gè)堿基C且無(wú)雜峰;Western blot檢測(cè)純合型小鼠Cyp2el蛋白表達(dá)量遠(yuǎn)低于野生型小鼠;酶活性檢測(cè)可見(jiàn)純合型小鼠與酶自身性質(zhì)相關(guān)的Km值與野生型相差不大,與酶含量相關(guān)的Vmax遠(yuǎn)低于野生型小鼠;2、DMF致小鼠急性肝毒性效應(yīng):雌雄性野生型小鼠在染毒后三個(gè)時(shí)間點(diǎn)出現(xiàn)肝臟系數(shù)、肝酶ALT和AST水平顯著升高,純合型小鼠肝酶水平改變無(wú)統(tǒng)計(jì)學(xué)差異。野生型小鼠肝臟病理在染毒后出現(xiàn)肝細(xì)胞水樣變、大片性細(xì)胞壞死伴炎細(xì)胞浸潤(rùn),純合型小鼠在病變的范圍和程度上均較野生型小鼠輕;3、血紅蛋白加合物和CYP2E1:NMHb檢出有特異性,對(duì)照組不能檢出;雄性野生型小鼠暴露DMF后24 h已基本完成DMF代謝,生成NMHb;雌性野生型小鼠染毒后24 h時(shí)NMHb生成量較少,NMHb水平在48 h急劇升高,提示DMF代謝重要發(fā)生在24-48 h;純合型雌雄性小鼠均有少量NMHb生成。PCR檢測(cè)小鼠Cyp2e1mRNA水平可見(jiàn)染毒后有mRNA水平變化,Western blot實(shí)驗(yàn)未見(jiàn)純合型組明顯Cyp2el蛋白條帶,野生型小鼠Cyp2e1蛋白在染毒后有先升高后降低的趨勢(shì),提示DMF可能引起CYP2E1轉(zhuǎn)錄和翻譯水平的調(diào)控,并可能出現(xiàn)CYP2E1自身抑制;雌雄性野生型小鼠在染毒后48h出現(xiàn)Cyp2e1酶活性下降,提示可能與自身抑制有關(guān);雄性純合型小鼠酶活性本底值較低,染毒后有升高趨勢(shì);4、DMF所致氧化應(yīng)激和內(nèi)質(zhì)網(wǎng)應(yīng)激:DMF暴露會(huì)導(dǎo)致肝臟內(nèi)GSH含量降低,MDA含量升高,激活體內(nèi)SOD和GSH-Px等抗氧化酶的活性。內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP94和GRP78等蛋白水平在DMF暴露后升高;5、性別差異:血生化結(jié)果中雌性野生型小鼠在DMF染毒后出現(xiàn)甘油三酯和膽固醇的升高,而在雄性野生型小鼠中未見(jiàn)兩項(xiàng)指標(biāo)的改變。野生型小鼠因壞死明顯而肝臟病理組織學(xué)檢測(cè)未見(jiàn)性別差異,但在純合型小鼠中差異明顯,表現(xiàn)為雄性純合型小鼠病理組織學(xué)改變以肝細(xì)胞腫脹為主,雌性純合型小鼠病理組織學(xué)改變以肝臟脂肪變性為主。NMHb觀察到DMF的代謝情況,雄性野生型小鼠的DMF代謝主要發(fā)生在0-24 h,雌性野生型小鼠DMF代謝主要發(fā)生在24-48 h;結(jié)論1、成功構(gòu)建Cyp2e1基因小鼠純合型;2、CYP2E1在DMF致小鼠急性肝毒性效應(yīng)中起到了毒物的代謝活化、產(chǎn)生活性氧等重要作用;3、內(nèi)質(zhì)網(wǎng)應(yīng)激為可能的DMF致小鼠肝臟損傷的機(jī)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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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