本文選題：脫氧雪腐鐮刀菌烯醇 + 胚胎毒性�。� 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：脫氧雪腐鐮刀菌烯醇(Deoxynivalenol,DON)是糧谷類食物重要的污染物之一,其在大麥、小麥、玉米以及其他谷類作物中含量最高,而農(nóng)副產(chǎn)品(如啤酒、面包、雞蛋、牛奶和肉類等)中也有檢出。DON是單端孢霉烯族化合物B型中的一員,該族化合物已被證實由食物與環(huán)境中的鐮刀菌、葡萄穗霉菌以及漆斑菌等在生長過程中產(chǎn)生,并且其引起的食物中毒事件一直是全球食品安全工作者關(guān)注的重點之一。DON很難被一般的烹調(diào)和加熱降解,即其毒性在一般情況下很難被破壞,因此其在食品和環(huán)境中具有極高的殘留濃度,一旦形成將通過食物鏈的傳遞對人體和動物的健康產(chǎn)生巨大威脅。就目前的研究來看,DON可在多個物種體內(nèi)引起多器官的損傷,主要表現(xiàn)為惡心、嘔吐、拒食、腹瀉、消化紊亂以及體重減輕等相對較輕微的反應(yīng),也可引起皮膚損傷,免疫抑制甚至骨髓破壞等嚴(yán)重不良反應(yīng)。從另一方面來說,DON在體內(nèi)無特殊的靶器官,但具有很強的細(xì)胞毒性。同時DON具有多種生物毒作用,主要包括細(xì)胞毒性,免疫毒性,遺傳毒性,以及致畸性和致癌性等。近年來在體內(nèi)和體外模型上,DON被發(fā)現(xiàn)具有影響不同動物和細(xì)胞繁殖和胚胎發(fā)育的特征,豬和鼠是應(yīng)用廣泛的體內(nèi)實驗研究對象,而BeWo細(xì)胞和卵母細(xì)胞則是重要的體外實驗研究對象。雖然DON的胚胎發(fā)育毒性已被眾多研究所證實,但是其毒性作用機制仍需更深入的探討,而這一點將是本研究的重點之一。早期的實驗已證實細(xì)胞內(nèi)ROS過度堆積所引起的氧化應(yīng)激是DON引起早期毒作用的重要機制之一。本研究將關(guān)注該毒作用機制是否存在于DON誘導(dǎo)胎盤氧化損傷的模型中。胎盤(placenta)是母體與胎兒進行物質(zhì)交換的器官,胎兒在子宮中僅能依靠胎盤從母體獲得營養(yǎng)。HO-1是血紅素代謝的關(guān)鍵酶,其表達(dá)上調(diào)對于細(xì)胞來說是自我保護的適應(yīng)性機制之一,即一定程度上保護細(xì)胞免于各種因素引起的氧化損傷。就本實驗來說,一方面HO-1廣泛分布于全身(包括子宮和胎盤)組織細(xì)胞的微粒體內(nèi),能被多種刺激因素(如低氧狀態(tài)、內(nèi)毒素及紫外線照射等)引起的氧化應(yīng)激誘導(dǎo)表達(dá),這是機體抵抗氧化損傷的重要機制之一。另一方面HO-1的缺陷將導(dǎo)致多種妊娠不良結(jié)局的出現(xiàn)(如復(fù)發(fā)性流產(chǎn)、宮內(nèi)發(fā)育遲緩、死胎和先兆子癇等)。因此,我們認(rèn)為HO-1可作為本研究的效應(yīng)標(biāo)志物,同時探討其相關(guān)信號通路,即Nrf2/HO-1信號轉(zhuǎn)導(dǎo)通路,在該過程中的表達(dá)情況也是本研究的關(guān)注點之一。綜上所述,本課題一方面可確證DON所致胚胎發(fā)育毒性,另一方面將深入分析DON引起胚胎發(fā)育毒性的分子機制。因此,該課題的完成將對DON發(fā)育毒性的早期預(yù)警和風(fēng)險評估基礎(chǔ)資料的建立起到不可或缺的作用。第一部分妊娠期脫氧雪腐鐮刀菌烯醇暴露對C57BL/6小鼠胚胎毒性研究目的：對妊娠期DON染毒C57BL/6小鼠在妊娠末期進行胚胎發(fā)育評價,確證DON具有胚胎毒性,同時初步分析DON致胚胎毒性的特點。方法：小鼠適應(yīng)性喂養(yǎng)1w后以2：1的比例將雌雄小鼠合籠,次日以查見陰栓者(查出精子)定為妊娠0.5 d (Gestation day 0.5 d, GD0.5 d)。按體重將懷孕母鼠隨機分為4組后在母鼠GD9.5-11.5 d分別給予不同劑量DON溶液連續(xù)灌胃染毒。具體分組情況如下：(1)陰性對照組(Control):超純水；(2)DON低劑量組(DON-L):1.0 mg/kg/day;(3)DON中劑量組(DON-M):2.5 mg/kg/day;(4)DON高劑量組(DON-H): 5.0 mg/kg/day oGD18.5 d將母鼠頸椎脫臼處死,分離子宮觀察胎盤和胚胎。觀察外形的同時進行骨骼和內(nèi)臟的染色以確定是否存在相應(yīng)畸形,同時計算與胚胎發(fā)育相關(guān)各指標(biāo)的數(shù)值并進行統(tǒng)計分析。結(jié)果： (1)妊娠期DON暴露對母鼠的影響：DON對妊娠期母鼠引起的拒食和體重減輕等作用不明顯。各組母鼠的胎窩總重,胎盤總重和平均胎盤重未出現(xiàn)顯著性差異(P0.05);(2)妊娠期DON暴露對胚胎的影響：高劑量組的活胎率和吸收胎率與對照組相比具有顯著性差異(P0.01),且高劑量組多只母鼠均出現(xiàn)全吸收胎的情況。DON對小鼠胚胎發(fā)育的影響可能并不表現(xiàn)在外觀和內(nèi)臟畸形方面,而是表現(xiàn)在骨骼畸形方面,且全身各處骨骼均可檢出明顯的畸形。結(jié)論：(1)初步證實DON具有胚胎毒性,且GD9.5-11.5 d時5.0 mg/kg DON暴露主要導(dǎo)致吸收胎,而2.5 mg/kg DON暴露對胚胎的影響則主要表現(xiàn)為骨骼畸形,另外1.0mg/kg DON對母鼠和胚胎的影響未被觀察到：(2)DON暴露引起的骨骼畸形分布于全身,如頭頸部,中軸骨,鎖骨和四肢骨骼均可檢出明顯的畸形,而其中尾部畸形(卷尾)的發(fā)生率最高。第二部分妊娠期脫氧雪腐鐮刀菌烯醇暴露對C57BL/6小鼠胎盤氧化應(yīng)激的影響目的：觀察妊娠期DON暴露時胎盤氧化應(yīng)激水平和Nrf2/HO-1信號轉(zhuǎn)導(dǎo)通路表達(dá)水平。探討DON誘導(dǎo)胎盤氧化應(yīng)激在DON所致胚胎毒性中的角色,同時初步探討HO-1在此過程中所起作用。方法：小鼠適應(yīng)性喂養(yǎng)1 w后以2：1的比例將雌雄小鼠合籠,次日以查見陰栓者(查出精子)定為GD0.5 d。按體重將懷孕母鼠隨機分為4組后在母鼠GD9.5-11.5 d分別給予不同劑量DON溶液連續(xù)灌胃染毒。具體分組情況如下：(1)陰性對照組(Control):超純水；(2)DON低劑量組(DON-L):1.0 mg/kg/day;(3)DON中劑量組(DON-M):2.5 mg/kg/day;(4)DON高劑量組(DON-H):5.0 mg/kg/day。GD18.5 d將母鼠頸椎脫臼處死后進行后續(xù)實驗。將胎盤組織固定并進行HE染色,檢測胎盤組織ROS水平的同時對氧化和抗氧化指標(biāo)(MDA, SOD, GSH)水平進行分析。此外,PCR和WB檢測信號轉(zhuǎn)導(dǎo)通路Nrf2/HO-1相關(guān)基因mRNA和蛋白表達(dá)水平的同時使用ELISA的方法測定胎盤組織中HO-1的酶水平。另外,采用激光共聚焦法來測定細(xì)胞中Nrf2的轉(zhuǎn)位。最后,初步探討炎癥和凋亡相關(guān)基因在此過程中的表達(dá)水平。結(jié)果：(1)DON對胎盤的結(jié)構(gòu)和功能均具有損傷作用,在中劑量和高劑量組尤為明顯：(2)妊娠期DON暴露會引起胎盤ROS聚集,該現(xiàn)象在DON劑量為5.0 mg/kg/day時較為明顯。高劑量組MDA水平顯著高于對照組(P0.01)。低、中劑量組與對照組相比SOD水平顯著性上升(P0.05),且具有劑量效應(yīng),而高劑量組未出現(xiàn)顯著性改變。然而,3個DON處理組與對照組相比GSH水平均無顯著性差異；(3)3個DON處理組PIGF的mRNA表達(dá)水平均顯著性低于對照組(P0.05),而以上3組的蛋白質(zhì)水平雖均低于對照組,但僅高劑量組與對照組相比差異具有顯著性(P0.01)：(4)3個DON處理組HO-1酶水平均低于對照組,但從低劑量組到高劑量組表現(xiàn)出上調(diào)趨勢,其中低劑量組和中劑量組與對照組相比差異具有顯著性(P0.05);(5)HO-1的mRNA在低劑量組顯著性高于對照組(P0.05),而高劑量組顯著性低于對照組(P0.01),而Nrf2的mRNA在低劑量組和中劑量組均顯著性高于對照組(P0.05)。HO-1的蛋白表達(dá)在中劑量組(P0.01)和高劑量組(P0.05)均顯著性高于對照組,而Nrf2的蛋白表達(dá)在低劑量組(P0.05)、中劑量組(P0.05)和高劑量組(P0.01)均顯著性高于對照組。Keap-1的蛋白表達(dá)未出現(xiàn)顯著性改變。同時在該過程中胎盤中Nrf2由細(xì)胞質(zhì)向細(xì)胞核中轉(zhuǎn)位的現(xiàn)象可被觀察到；(6)炎癥相關(guān)指標(biāo)IL-6,IL-8,TNF-α和Cox-2的mRNA表達(dá)在3個DON處理組表現(xiàn)得頗為復(fù)雜,具體情況為低劑量組中發(fā)現(xiàn)TNF-α表達(dá)顯著性上調(diào)(P0.05),中劑量組Cox-2表達(dá)顯著性下調(diào)(P0.05),而高劑量組則是IL-6顯著性下調(diào)(P0.05)而IL-8顯著性上調(diào)(P0.05)。結(jié)論：(1)妊娠期母鼠DON暴露將對母鼠胎盤產(chǎn)生結(jié)構(gòu)和功能上的雙重?fù)p傷;(2)胎盤結(jié)構(gòu)和功能的損傷可能與DON引起的胎盤氧化應(yīng)激相關(guān),炎癥和凋亡也在該過程中出現(xiàn),但其具體變化情況仍需進一步研究；(3)胎盤中Nrf2/HO-1信號轉(zhuǎn)導(dǎo)通路在DON發(fā)揮胚胎毒性過程中發(fā)生表達(dá)改變,并可能起到保護胎盤和胚胎發(fā)育的作用。第三部分脫氧雪腐鐮刀菌烯醇染毒對BeWo細(xì)胞毒性分子機制研究目的：對BeWo細(xì)胞給予不同劑量的DON染毒,同時結(jié)合HO-1激活劑(Hemin)和抑制劑(Znpp),探討DON染毒導(dǎo)致的氧化應(yīng)激反應(yīng)對該細(xì)胞的影響。另外,驗證HO-1在DON染毒時在BeWo細(xì)胞中表達(dá)的時間-和劑量-效應(yīng)關(guān)系。方法：復(fù)蘇細(xì)胞后在培養(yǎng)基中進行培養(yǎng),每日觀察細(xì)胞的生長和貼壁狀況,當(dāng)細(xì)胞融合程度達(dá)到80%時即可進行細(xì)胞傳代。而后按實驗要求將細(xì)胞接種到所需器皿上(如12孔板,96孔板或培養(yǎng)皿等),接種后保持隔天換液。使用CCK-8試劑盒進行細(xì)胞活力檢測,確定具體的分組：(1)陰性對照組：0 nM DON;(2)DON處理組：50 nM DON;(3)HO-1激活劑組：50 nM DON+10μM Hemin;(4)HO-1抑制劑組：50 nM DON+10μM Znpp。按以上分組培養(yǎng)1 h(僅PCR),3 h,12 h和24 h后進行后續(xù)實驗。使用ELISA試劑盒分別測定細(xì)胞分泌P-HCG的水平和HO-1的酶水平。多功能酶標(biāo)儀被用來測定細(xì)胞內(nèi)ROS水平,此外運用不同的試劑盒測定細(xì)胞中MDA, SOD和GSH水平。PCR檢測信號轉(zhuǎn)導(dǎo)通路Nrf2/HO-1相關(guān)基因mRNA的表達(dá)水平。另外,采用激光共聚焦法來測定細(xì)胞中Nrf2的轉(zhuǎn)位。最后,PCR初步分析炎癥和凋亡相關(guān)基因mRNA在此過程中的表達(dá)水平。結(jié)果： (1)所有DON處理組P-HCG水平均顯著性低于對照組(P0.01),在3h和12 h時HO-1激活劑和抑制劑組與50 nM DON處理組相比均無顯著性差異,而在24 h時以上兩組P-HCG水平均顯著性低于50 nM DON處理組(P0.05)；(2)DON處理時細(xì)胞發(fā)生顯著的ROS堆積(P0.01),而HO-1激活劑能顯著性改善DON處理引起的ROS堆積(P0.01)。3h時HO-1激活劑和抑制劑組MDA水平均顯著性高于DON處理組(P0.01),但在12h時HO-1激活劑組變?yōu)轱@著性低于DON處理組(P0.05)。Hemin在3h表現(xiàn)為升高SOD水平(P0.05),但此后均顯著性下調(diào)SOD水平(P0.01),而Znpp僅在12h表現(xiàn)為顯著性上調(diào)其水平(P0.01)。Hemin在3h顯著性下調(diào)GSH水平(P0.01),而Znpp則在24 h顯著性上調(diào)GSH水平(P0.01)；(3)3 h時Hemin和Znpp分別表現(xiàn)出相應(yīng)的上調(diào)和下調(diào)HO-1酶水平的作用(P0.01),12h時3個DON處理組無顯著性差異,24 h時Hemin和Znpp均表現(xiàn)為上調(diào)HO-1水平(P0.01)；(4)Hemin和Znpp在各時間點均升高HO-1的mRNA表達(dá)水平(P0.01),Hemin僅上調(diào)3 h時Nrf2的表達(dá)(P0.05),而Znpp上調(diào)12 h和24 h時Nrf2的mRNA表達(dá)(P0.01)。此外,Hemin和Znpp預(yù)處理的BeWo細(xì)胞在較長時間DON處理(如12 h和24 h)時,Nrf2將出現(xiàn)明顯的從細(xì)胞質(zhì)向細(xì)胞核轉(zhuǎn)位的過程；(5)隨著DON處理時間和劑量的增加,細(xì)胞炎癥反應(yīng)不斷增強,各炎癥因子表達(dá)趨勢較為復(fù)雜。結(jié)論： (1)早期DON處理時,HO-1激活劑改善DON導(dǎo)致的細(xì)胞功能障礙,而隨著時間的進展,HO-1激活劑的該功能逐漸失效,反而是HO-1抑制劑起到更有效的改善功效;(2)ROS水平對DON染毒時的胎盤滋養(yǎng)層細(xì)胞中HO-1的表達(dá)存在時間-和劑量-效應(yīng)關(guān)系；(3)胎盤氧化應(yīng)激是DON產(chǎn)生胚胎毒性的機制之一,激活HO-1的活性在早期可上調(diào)細(xì)胞的抗氧化能力,但隨著時間的推移該作用逐漸消失,甚至HO-1過度激活將會加重細(xì)胞的氧化損傷,即HO-1對氧化應(yīng)激的保護作用存在“閾值效應(yīng)”。
[Abstract]:Deoxynivalenol (DON) is one of the most important contaminants in cereals. It has the highest content in barley, wheat, corn, and other cereals, and a member of the agricultural and sideline products (such as beer, bread, eggs, milk and meat) is also found to be a member of the B type of monosamycin compound. The compound has been found. It is proved that Fusarium, panicle and lacquer fungus in food and environment are produced during the growth process, and the food poisoning caused by it has always been one of the focus of food safety workers worldwide..DON is difficult to be cooked and heated by general cooking, that is, its toxicity is difficult to be destroyed in general, so it is in food. In the present study, DON can cause multiple organ damage in many species, mainly as nausea, vomiting, antifeedant, diarrhoea, digestive disorders and weight loss. Reaction can also cause serious adverse reactions such as skin damage, immunosuppression and even bone marrow destruction. On the other hand, DON has no special target organs in the body, but has a strong cytotoxicity. At the same time, DON has a variety of biological toxicity, mainly including cytotoxicity, immunotoxicity, genetic toxicity, teratogenicity and carcinogenicity. In vivo and in vitro models, DON has been found to have the characteristics that affect the reproduction and embryonic development of different animals and cells. Pigs and mice are widely used in vivo experiments, while BeWo and oocytes are important in vitro experimental research. Although the embryonic developmental toxicity of DON has been confirmed by many studies, the toxicity of it has been proved to be toxic. This study will be one of the key points in this study. Early experiments have confirmed that oxidative stress caused by overaccumulation of ROS in cells is one of the most important mechanisms for early toxic effects of DON. This study will focus on whether the mechanism exists in the model of DON induced oxidative damage in the placenta. Placenta) is the organ exchange between the mother and the fetus, and the fetus in the uterus can only rely on the placenta to obtain nutrient.HO-1 from the mother body as the key enzyme of heme metabolism. Its expression is one of the adaptive mechanisms for the self-protection of the cells, that is, to some extent, protect the cells from various factors caused by oxidative damage. On the one hand, HO-1 is widely distributed in the body of the body (including the uterus and placenta) tissue cells, which can be induced by a variety of stimulant factors (such as hypoxia, endotoxin and ultraviolet radiation) induced by oxidative stress, which is one of the important mechanisms of the body to resist oxidative damage. On the other hand, the defects of HO-1 will lead to a variety of pregnancy induced pregnancy. The occurrence of adverse outcomes (such as recurrent abortion, intrauterine retardation, stillbirth and preeclampsia). Therefore, we think that HO-1 can be used as a marker for this study and to explore its related signaling pathways, that is, the Nrf2/HO-1 signal transduction pathway, and the expression in this process is also one of the concerns of this study. On the one hand, it can confirm the developmental toxicity of DON induced embryo. On the other hand, it will analyze the molecular mechanism of embryonic development toxicity caused by DON. Therefore, the completion of this topic will play an indispensable role in the early warning of developmental toxicity of DON and the establishment of the basic data of risk assessment. The aim of C57BL/6 mouse embryo toxicity study was to evaluate the embryo development of C57BL/6 mice infected by DON in pregnancy at the end of pregnancy. It was confirmed that DON had embryonic toxicity, and the characteristics of embryo toxicity caused by DON were preliminarily analyzed. Methods: the mice were fed to 1W in the proportion of 2:1 in the mice after adaptive feeding, and the following day was found to find the suppositories. The pregnancy was determined to be 0.5 D of pregnancy (Gestation day 0.5 D, GD0.5 d). The pregnant female rats were randomly divided into 4 groups according to weight, and the GD9.5-11.5 D of the female rats was given the different doses of DON solution for continuous gavage. The specific grouping conditions were as follows: (1) negative control group (Control): ultra pure water; (2) DON low dose group (DON-L): 1 M): 2.5 mg/kg/day; (4) DON high dose group (DON-H): 5 mg/kg/day oGD18.5 d to dislocate the cervical vertebra of the mother rat, separate the uterus and observe the placenta and embryo. Observe the appearance of the bone and viscera to determine whether there is a corresponding malformation, and calculate the values of the index of the embryo development and the statistical analysis. Results: (1 The effect of DON exposure during pregnancy on the female rats: the effect of DON on antifeedant and weight loss caused by pregnant female rats was not obvious. There was no significant difference between the total weight of the fetal nests, the total placenta weight and the average placental weight (P0.05); (2) the effect of DON exposure during pregnancy on the embryo: the rate of live fetal and the absorption rate of the high dose group compared with the control group. There was significant difference (P0.01), and in the high dose group, all the female mice had full absorption fetus. The effect of.DON on the development of mouse embryo may not be manifested in appearance and visceral malformation, but in skeletal deformity, and all the bones in all parts of the body can detect obvious deformities. Conclusion: (1) it is preliminarily confirmed that DON has embryonic toxicity, And the exposure of 5 mg/kg DON at GD9.5-11.5 D mainly leads to the absorption of fetus, while the effect of 2.5 mg/kg DON exposure on the embryo is mainly manifested as bone malformation, and the effect of 1.0mg/kg DON on mother and embryo is not observed: (2) the skeletal deformity caused by DON exposure is distributed throughout the body, such as the head and neck, the middle axis bone, clavicle, and extremities can be detected. Obviously deformity, and the incidence of tail malformation (coiling) is the highest. The effect of second partial pregnancy deoxyfusarium exposure on oxidative stress in C57BL/6 mouse placenta: To observe the placental oxidative stress level and the expression level of Nrf2/HO-1 signal transduction pathway during DON exposure during pregnancy. To explore the oxidative stress induced by DON in placenta The role of DON in embryo toxicity and the role of HO-1 in this process was preliminarily discussed. Method: after 1 W of adaptive feeding in mice, the female and male mice were caged with the proportion of 2:1. The following day, the female mice were determined to be GD0.5 D., and the pregnant female rats were randomly divided into 4 groups according to the weight, and the female mice GD9.5-11.5 D were given different agents respectively. DON solution was administered continuously. The specific grouping conditions were as follows: (1) negative control group (Control): ultra pure water; (2) DON low dose group (DON-L): 1 mg/kg/day; (3) DON medium dose group (DON-M): 2.5 mg/kg/day; (4) DON high dose group (DON-H): 5 mg/ kg/day.GD18.5, after the dislocation of the cervical vertebra of the mother rat followed up experiment. Placental tissue fixed and HE staining was carried out to detect the level of ROS in placenta tissue and the levels of oxidation and antioxidant index (MDA, SOD, GSH) were analyzed. In addition, PCR and WB were used to detect the mRNA and protein expression level of Nrf2/HO-1 related genes in the signal transduction pathway, while ELISA method was used to determine the HO-1 enzyme level in the placental fabric. In addition, the laser confocal method was used. Determine the transposition of Nrf2 in the cells. Finally, we preliminarily discuss the expression level of inflammation and apoptosis related genes in this process. Results: (1) DON has a damaging effect on the structure and function of the placenta, especially in the medium dose and high dose group: (2) the DON exposure in pregnancy will cause the ROS aggregation in the placenta, which is at the dosage of 5 mg/kg/day at DON. The level of MDA in high dose group was significantly higher than that of the control group (P0.01). The level of SOD in the middle dose group was significantly higher than that of the control group (P0.05), and there was a dose effect, but there was no significant change in the high dose group. However, there was no significant difference in the GSH water between the 3 DON treatment groups and the control group; (3) the mRNA table of PIGF in the 3 DON treatment group. The average value of water content was lower than that of the control group (P0.05), while the protein levels in the above 3 groups were lower than the control group, but the difference in the high dose group was significantly higher than the control group (P0.01): (4) the level of HO-1 enzyme in the 3 DON treatment groups was lower than the control group, but the low dose group to the high dose group showed the up-regulation trend, of which the low dose group and the medium agent were in the low dose group. The difference between the group and the control group was significant (P0.05); (5) the significance of mRNA in the low dose group was significantly higher than that in the control group (P0.05), while the high dose group was significantly lower than the control group (P0.01), while the Nrf2 mRNA in the low dose group and the middle dose group were significantly higher than the control group (P0.05).HO-1 protein expression in the middle dose group (P0.01) and the high dose group (P0 (P0). .05) was significantly higher than that in the control group, while the protein expression of Nrf2 was in the low dose group (P0.05). Both the medium dose group (P0.05) and the high dose group (P0.01) were significantly higher than the.Keap-1 in the control group. The phenomenon of the transposition of the Nrf2 from the cytoplasm to the nucleus in the placenta could be observed; (6) the inflammatory phase was observed. The mRNA expression of IL-6, IL-8, TNF- a, and Cox-2 in the 3 DON treatment groups was quite complex. The specific situation was that the expression of TNF- a was significantly up (P0.05) in the low dose group and the significant downregulation of Cox-2 expression in the middle dose group (P0.05), while the high dose group was IL-6 significant downregulation (P0.05). (1) pregnancy DON exposure in the stage of the mother mouse produces double damage to the structure and function of the placenta of the mother mouse. (2) the damage of the placental structure and function may be related to the oxidative stress caused by DON. Inflammation and apoptosis also appear in this process, but the specific changes still need to be further studied. (3) the Nrf2/HO-1 signal transduction pathway in the placenta plays the embryo in DON. The expression changes in the process of fetal toxicity and the role of protecting the placenta and embryo development. Third the study of the molecular mechanism of BeWo cell toxicity by deoxynivalenol: BeWo cells were exposed to different doses of DON, combined with HO-1 activator (Hemin) and inhibitor (Znpp), to explore the result of DON poisoning. The effect of oxidative stress reaction on the cell. In addition, the time and dose effect relationship of HO-1 in BeWo cells was verified by DON. Methods: after the cell was resuscitated, the cells were cultured in the medium, and the cell growth and adherence were observed daily. The cell passage could be carried out when the degree of cell fusion reached 80%. The cells were inoculated to the required vessels (such as 12 orifice plates, 96 orifice plates or culture Petri dishes) to keep the liquid after inoculation. Cell viability was detected by CCK-8 kits, and specific groups were used to determine specific groups: (1) negative control groups: 0 nM DON; (2) DON treatment group: 50 nM DON; (3) HO-1 activator group: 50 nM DON+10 M Hemin; (4) 50 HO-1 inhibitors: 50 HO-1 inhibitor groups 50 50 M DON+10 mu M Znpp. was used to train 1 h (only PCR), 3 h, 12 h and 24 h for subsequent experiments. A ELISA reagent kit was used to determine the level of cell secretion and the level of HO-1 enzymes. The expression level of the Nrf2/HO-1 related gene mRNA in the transduction pathway. In addition, the translocation of Nrf2 in cells was measured by laser confocal method. Finally, the expression level of inflammation and apoptosis related gene mRNA in this process was preliminarily analyzed. Results: (1) all the P-HCG levels in all DON treatment groups were significantly lower than that of the control group (P0.01) and HO-1 irritable at 3H and 12 h. There was no significant difference between the active agent and the inhibitor group and the 50 nM DON treatment group, while the two groups of P-HCG levels above the 24 h were significantly lower than the 50 nM DON treatment group (P0.05). (2) the ROS accumulation (P0.01) occurred during the DON treatment, and the HO-1 activator could significantly improve the activator and inhibition when the DON process caused the accumulation. The level of MDA in the preparation group was significantly higher than that in the DON treatment group (P0.01), but at 12h, the HO-1 activator group became significantly lower than the DON treatment group (P0.05).Hemin in 3H performance increased SOD level (P0.05). Level (P0.01), while Znpp significantly increased GSH level at 24 h (P0.01); (3) Hemin and Znpp showed corresponding up and down at 3 h respectively.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R114

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