脱氧雪腐镰刀菌烯醇的胚胎毒性及胎盘氧化应激所起作用

发布时间：2018-06-16 06:07

本文选题：脱氧雪腐镰刀菌烯醇 + 胚胎毒性　；参考：《华中科技大学》2016年博士论文

【摘要】：脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)是粮谷类食物重要的污染物之一,其在大麦、小麦、玉米以及其他谷类作物中含量最高,而农副产品(如啤酒、面包、鸡蛋、牛奶和肉类等)中也有检出。DON是单端孢霉烯族化合物B型中的一员,该族化合物已被证实由食物与环境中的镰刀菌、葡萄穗霉菌以及漆斑菌等在生长过程中产生,并且其引起的食物中毒事件一直是全球食品安全工作者关注的重点之一。DON很难被一般的烹调和加热降解,即其毒性在一般情况下很难被破坏,因此其在食品和环境中具有极高的残留浓度,一旦形成将通过食物链的传递对人体和动物的健康产生巨大威胁。就目前的研究来看,DON可在多个物种体内引起多器官的损伤,主要表现为恶心、呕吐、拒食、腹泻、消化紊乱以及体重减轻等相对较轻微的反应,也可引起皮肤损伤,免疫抑制甚至骨髓破坏等严重不良反应。从另一方面来说,DON在体内无特殊的靶器官,但具有很强的细胞毒性。同时DON具有多种生物毒作用,主要包括细胞毒性,免疫毒性,遗传毒性,以及致畸性和致癌性等。近年来在体内和体外模型上,DON被发现具有影响不同动物和细胞繁殖和胚胎发育的特征,猪和鼠是应用广泛的体内实验研究对象,而BeWo细胞和卵母细胞则是重要的体外实验研究对象。虽然DON的胚胎发育毒性已被众多研究所证实,但是其毒性作用机制仍需更深入的探讨,而这一点将是本研究的重点之一。早期的实验已证实细胞内ROS过度堆积所引起的氧化应激是DON引起早期毒作用的重要机制之一。本研究将关注该毒作用机制是否存在于DON诱导胎盘氧化损伤的模型中。胎盘(placenta)是母体与胎儿进行物质交换的器官,胎儿在子宫中仅能依靠胎盘从母体获得营养。HO-1是血红素代谢的关键酶,其表达上调对于细胞来说是自我保护的适应性机制之一,即一定程度上保护细胞免于各种因素引起的氧化损伤。就本实验来说,一方面HO-1广泛分布于全身(包括子宫和胎盘)组织细胞的微粒体内,能被多种刺激因素(如低氧状态、内毒素及紫外线照射等)引起的氧化应激诱导表达,这是机体抵抗氧化损伤的重要机制之一。另一方面HO-1的缺陷将导致多种妊娠不良结局的出现(如复发性流产、宫内发育迟缓、死胎和先兆子痫等)。因此,我们认为HO-1可作为本研究的效应标志物,同时探讨其相关信号通路,即Nrf2/HO-1信号转导通路,在该过程中的表达情况也是本研究的关注点之一。综上所述,本课题一方面可确证DON所致胚胎发育毒性,另一方面将深入分析DON引起胚胎发育毒性的分子机制。因此,该课题的完成将对DON发育毒性的早期预警和风险评估基础资料的建立起到不可或缺的作用。第一部分妊娠期脱氧雪腐镰刀菌烯醇暴露对C57BL/6小鼠胚胎毒性研究目的：对妊娠期DON染毒C57BL/6小鼠在妊娠末期进行胚胎发育评价,确证DON具有胚胎毒性,同时初步分析DON致胚胎毒性的特点。方法：小鼠适应性喂养1w后以2：1的比例将雌雄小鼠合笼,次日以查见阴栓者(查出精子)定为妊娠0.5 d (Gestation day 0.5 d, GD0.5 d)。按体重将怀孕母鼠随机分为4组后在母鼠GD9.5-11.5 d分别给予不同剂量DON溶液连续灌胃染毒。具体分组情况如下：(1)阴性对照组(Control):超纯水；(2)DON低剂量组(DON-L):1.0 mg/kg/day;(3)DON中剂量组(DON-M):2.5 mg/kg/day;(4)DON高剂量组(DON-H): 5.0 mg/kg/day oGD18.5 d将母鼠颈椎脱臼处死,分离子宫观察胎盘和胚胎。观察外形的同时进行骨骼和内脏的染色以确定是否存在相应畸形,同时计算与胚胎发育相关各指标的数值并进行统计分析。结果： (1)妊娠期DON暴露对母鼠的影响：DON对妊娠期母鼠引起的拒食和体重减轻等作用不明显。各组母鼠的胎窝总重,胎盘总重和平均胎盘重未出现显著性差异(P0.05);(2)妊娠期DON暴露对胚胎的影响：高剂量组的活胎率和吸收胎率与对照组相比具有显著性差异(P0.01),且高剂量组多只母鼠均出现全吸收胎的情况。DON对小鼠胚胎发育的影响可能并不表现在外观和内脏畸形方面,而是表现在骨骼畸形方面,且全身各处骨骼均可检出明显的畸形。结论：(1)初步证实DON具有胚胎毒性,且GD9.5-11.5 d时5.0 mg/kg DON暴露主要导致吸收胎,而2.5 mg/kg DON暴露对胚胎的影响则主要表现为骨骼畸形,另外1.0mg/kg DON对母鼠和胚胎的影响未被观察到：(2)DON暴露引起的骨骼畸形分布于全身,如头颈部,中轴骨,锁骨和四肢骨骼均可检出明显的畸形,而其中尾部畸形(卷尾)的发生率最高。第二部分妊娠期脱氧雪腐镰刀菌烯醇暴露对C57BL/6小鼠胎盘氧化应激的影响目的：观察妊娠期DON暴露时胎盘氧化应激水平和Nrf2/HO-1信号转导通路表达水平。探讨DON诱导胎盘氧化应激在DON所致胚胎毒性中的角色,同时初步探讨HO-1在此过程中所起作用。方法：小鼠适应性喂养1 w后以2：1的比例将雌雄小鼠合笼,次日以查见阴栓者(查出精子)定为GD0.5 d。按体重将怀孕母鼠随机分为4组后在母鼠GD9.5-11.5 d分别给予不同剂量DON溶液连续灌胃染毒。具体分组情况如下：(1)阴性对照组(Control):超纯水；(2)DON低剂量组(DON-L):1.0 mg/kg/day;(3)DON中剂量组(DON-M):2.5 mg/kg/day;(4)DON高剂量组(DON-H):5.0 mg/kg/day。GD18.5 d将母鼠颈椎脱臼处死后进行后续实验。将胎盘组织固定并进行HE染色,检测胎盘组织ROS水平的同时对氧化和抗氧化指标(MDA, SOD, GSH)水平进行分析。此外,PCR和WB检测信号转导通路Nrf2/HO-1相关基因mRNA和蛋白表达水平的同时使用ELISA的方法测定胎盘组织中HO-1的酶水平。另外,采用激光共聚焦法来测定细胞中Nrf2的转位。最后,初步探讨炎症和凋亡相关基因在此过程中的表达水平。结果：(1)DON对胎盘的结构和功能均具有损伤作用,在中剂量和高剂量组尤为明显：(2)妊娠期DON暴露会引起胎盘ROS聚集,该现象在DON剂量为5.0 mg/kg/day时较为明显。高剂量组MDA水平显著高于对照组(P0.01)。低、中剂量组与对照组相比SOD水平显著性上升(P0.05),且具有剂量效应,而高剂量组未出现显著性改变。然而,3个DON处理组与对照组相比GSH水平均无显著性差异；(3)3个DON处理组PIGF的mRNA表达水平均显著性低于对照组(P0.05),而以上3组的蛋白质水平虽均低于对照组,但仅高剂量组与对照组相比差异具有显著性(P0.01)：(4)3个DON处理组HO-1酶水平均低于对照组,但从低剂量组到高剂量组表现出上调趋势,其中低剂量组和中剂量组与对照组相比差异具有显著性(P0.05);(5)HO-1的mRNA在低剂量组显著性高于对照组(P0.05),而高剂量组显著性低于对照组(P0.01),而Nrf2的mRNA在低剂量组和中剂量组均显著性高于对照组(P0.05)。HO-1的蛋白表达在中剂量组(P0.01)和高剂量组(P0.05)均显著性高于对照组,而Nrf2的蛋白表达在低剂量组(P0.05)、中剂量组(P0.05)和高剂量组(P0.01)均显著性高于对照组。Keap-1的蛋白表达未出现显著性改变。同时在该过程中胎盘中Nrf2由细胞质向细胞核中转位的现象可被观察到；(6)炎症相关指标IL-6,IL-8,TNF-α和Cox-2的mRNA表达在3个DON处理组表现得颇为复杂,具体情况为低剂量组中发现TNF-α表达显著性上调(P0.05),中剂量组Cox-2表达显著性下调(P0.05),而高剂量组则是IL-6显著性下调(P0.05)而IL-8显著性上调(P0.05)。结论：(1)妊娠期母鼠DON暴露将对母鼠胎盘产生结构和功能上的双重损伤;(2)胎盘结构和功能的损伤可能与DON引起的胎盘氧化应激相关,炎症和凋亡也在该过程中出现,但其具体变化情况仍需进一步研究；(3)胎盘中Nrf2/HO-1信号转导通路在DON发挥胚胎毒性过程中发生表达改变,并可能起到保护胎盘和胚胎发育的作用。第三部分脱氧雪腐镰刀菌烯醇染毒对BeWo细胞毒性分子机制研究目的：对BeWo细胞给予不同剂量的DON染毒,同时结合HO-1激活剂(Hemin)和抑制剂(Znpp),探讨DON染毒导致的氧化应激反应对该细胞的影响。另外,验证HO-1在DON染毒时在BeWo细胞中表达的时间-和剂量-效应关系。方法：复苏细胞后在培养基中进行培养,每日观察细胞的生长和贴壁状况,当细胞融合程度达到80%时即可进行细胞传代。而后按实验要求将细胞接种到所需器皿上(如12孔板,96孔板或培养皿等),接种后保持隔天换液。使用CCK-8试剂盒进行细胞活力检测,确定具体的分组：(1)阴性对照组：0 nM DON;(2)DON处理组：50 nM DON;(3)HO-1激活剂组：50 nM DON+10μM Hemin;(4)HO-1抑制剂组：50 nM DON+10μM Znpp。按以上分组培养1 h(仅PCR),3 h,12 h和24 h后进行后续实验。使用ELISA试剂盒分别测定细胞分泌P-HCG的水平和HO-1的酶水平。多功能酶标仪被用来测定细胞内ROS水平,此外运用不同的试剂盒测定细胞中MDA, SOD和GSH水平。PCR检测信号转导通路Nrf2/HO-1相关基因mRNA的表达水平。另外,采用激光共聚焦法来测定细胞中Nrf2的转位。最后,PCR初步分析炎症和凋亡相关基因mRNA在此过程中的表达水平。结果： (1)所有DON处理组P-HCG水平均显著性低于对照组(P0.01),在3h和12 h时HO-1激活剂和抑制剂组与50 nM DON处理组相比均无显著性差异,而在24 h时以上两组P-HCG水平均显著性低于50 nM DON处理组(P0.05)；(2)DON处理时细胞发生显著的ROS堆积(P0.01),而HO-1激活剂能显著性改善DON处理引起的ROS堆积(P0.01)。3h时HO-1激活剂和抑制剂组MDA水平均显著性高于DON处理组(P0.01),但在12h时HO-1激活剂组变为显著性低于DON处理组(P0.05)。Hemin在3h表现为升高SOD水平(P0.05),但此后均显著性下调SOD水平(P0.01),而Znpp仅在12h表现为显著性上调其水平(P0.01)。Hemin在3h显著性下调GSH水平(P0.01),而Znpp则在24 h显著性上调GSH水平(P0.01)；(3)3 h时Hemin和Znpp分别表现出相应的上调和下调HO-1酶水平的作用(P0.01),12h时3个DON处理组无显著性差异,24 h时Hemin和Znpp均表现为上调HO-1水平(P0.01)；(4)Hemin和Znpp在各时间点均升高HO-1的mRNA表达水平(P0.01),Hemin仅上调3 h时Nrf2的表达(P0.05),而Znpp上调12 h和24 h时Nrf2的mRNA表达(P0.01)。此外,Hemin和Znpp预处理的BeWo细胞在较长时间DON处理(如12 h和24 h)时,Nrf2将出现明显的从细胞质向细胞核转位的过程；(5)随着DON处理时间和剂量的增加,细胞炎症反应不断增强,各炎症因子表达趋势较为复杂。结论： (1)早期DON处理时,HO-1激活剂改善DON导致的细胞功能障碍,而随着时间的进展,HO-1激活剂的该功能逐渐失效,反而是HO-1抑制剂起到更有效的改善功效;(2)ROS水平对DON染毒时的胎盘滋养层细胞中HO-1的表达存在时间-和剂量-效应关系；(3)胎盘氧化应激是DON产生胚胎毒性的机制之一,激活HO-1的活性在早期可上调细胞的抗氧化能力,但随着时间的推移该作用逐渐消失,甚至HO-1过度激活将会加重细胞的氧化损伤,即HO-1对氧化应激的保护作用存在“阈值效应”。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R114

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