无脊椎动物家蚕为材料的医学实验动物替代研究
发布时间：2018-07-16 08:07
【摘要】：实验动物替代方法,已广泛应用于生命科学研究领域。家蚕是鳞翅目模式昆虫,有着数千年畜牧化养殖历史,且已积累了系统深入的生理学、病理学和毒理学资料。家蚕遗传突变资源的创新及其在基础生命体系、物质代谢、能量代谢和遗传方式上与哺乳类有众多相似性的发现,使家蚕作为中国特色的模式昆虫正逐渐向实验动物模式化发展。然而,如何通过系统地控制实验用家蚕的遗传因素和生长环境,实现家蚕“实验动物化”,如何根据家蚕的生物学特性,优选出替代方法研究类型,是家蚕在成为标准的实验动物过程中所必须突破的瓶颈。另外,家蚕与小鼠等经典哺乳类模式动物相比,在外源化合物代谢动力学方面究竟存在哪些共性和差异,国内外也尚无系统的研究。本研究以家蚕实验动物化的标准建设为目的,总结了家蚕实验动物化的品种选育、饲养管理、设施管理等规范,设计了家蚕标准化饲养微屏障系统;通过肝毒性模式药物对乙酰氨基酚(APAP)处理,系统地比较研究家蚕与哺乳动物体内APAP的急性毒性分级和药代动力学特性;通过代谢组学方法、高通量分析(RNA-Seq)方法调查APAP对家蚕内源性代谢物和基因表达的差异,揭示APAP在体内所产生的生物效应;用试剂盒检测APAP染毒后家蚕催化药物代谢关键酶的活性,并通过生物信息学方法分析比较家蚕与小鼠等哺乳类在这些关键酶进化上的差异及活性位点差异。研究获得的主要结果如下:1.模药APAP对家蚕的急性毒性分级、药代动力学特征能够为医学实验动物替代建立平行数据急性毒性试验结果显示,APAP对家蚕毒性因品种不同而表现出毒性敏感性不同,大造较皓月敏感。APAP处理雌、雄大造品种的LD50分别为2017±254μg/g和2169±300μg/g。急性毒性分级为中低级毒性,与哺乳类相似。代谢动力学结果表明,低浓度600μg/g剂量和高浓度3600μg/g剂量APAP在家蚕体内的药动学呈一室开放型模型,消除半衰期(t1/2)分别为1.06h和2.77h;原药达峰时间(tmax)分别为0.50h和1.00h;原药达峰值浓度(Cmax)分别为67.69μg/ml和568.73μg/ml。相对生物利用度(AUC0-t)分别为:222.63 h?μg/ml和1976.32 h?μg/ml。代谢中间产物有N-乙酰对苯醌亚胺(NAPQI)产生,与哺乳类相比,药物吸收分布、体内代谢中间产物、总体的动力学(PK)相似,能为医学实验动物替代建立平行数据。2.模药APAP在家蚕的体内能产生与哺乳类共同的生物效应代谢组学分析显示,以600μg/g剂量APAP经口给药家蚕8 h后,循环血中差异代谢物在三羧酸循环、糖酵解途径、氨基酸代谢、黑色素生成等途径中,说明APAP在体内所产生的毒性途径主是因为药物代谢过程中产生了氧化应激,影响了体内能量供给、物质转运和信号传导。代谢物差异分析发现,APAP诱导家蚕血淋巴中氨基酸代谢产生差异,推测氨基酸代谢的异常和苹果酸、琥珀酸、延胡索酸等内源代谢物的显著下降相关,也与APAP染毒后产生的氧化应激相关。酪氨酸、多巴、海藻糖的显著升高进一步证实过量的APAP导致了氧化应激的产生;APAP诱导家蚕的胆固醇降低,却使β-羟基β-甲基戊二酸升高,表明APAP导致家蚕的胆固醇合成途径失衡,甾体激素的原料供给障碍影响机体的激素调节。3.模药APAP在家蚕的体内能产生与哺乳类共同的药理作用和毒性机理RNA-Seq结果分析APAP给药实验组与对照组的差异基因表达显示,在KEGG分析系统中与新陈代谢相关的通路里,大量的差异表达基因富集于三羧酸循环、氧化磷酸化、脂肪酸的氧化、糖酵解和糖异生等高产能环节,从基因表达水平上进一步证明,APAP对家蚕机体的伤害,主要是氧化应激和能量代谢、物质转运、信号传导等途径异常所至,这与哺乳类所产生的肝损伤机理一致。在KEGG分析系统中与有机体系统相关的通路里,大量的差异表达基因富集于心脏肌肉收缩、血管平滑肌收缩和调节肌动蛋白骨架等循环系统的相关通路,以及部分神经元突触基因通路,这些与代谢组学结果一致。推测APAP对家蚕具有与哺乳类相一致的抑制花生四烯酸通路的药理作用。4.家蚕与小鼠的药物代谢关键酶具有共同的活性位点和蛋白质互作机制生物信息分析结果显示,家蚕和小鼠的谷胱甘肽转硫酶家族成员(GSTs)在结构域和功能域方面,大多具有GSTs活性区域以及硫氧还蛋白的折叠模式;家蚕和小鼠的谷氨酸半胱氨酸连接酶催化亚基(GCLc)蛋白都具有谷胱甘肽合成酶的功能域,谷氨酸半胱氨酸连接酶(GCLM)都具有NADP依赖氧化还原酶功能域;家蚕硫氧还蛋白过氧化物酶家族(TPXs)中Jafrac1基因编码的过氧化物酶蛋白与小鼠的Prdx1和Prdx2基因编码的过氧化物酶具有高度保守的结构,家蚕的预测蛋白LOC733003、LOC101735759、LOC732921分别具有小鼠的Prdx3、Prdx4、Prdx5编码蛋白的保守结构域;家蚕的2个葡萄糖醛酸转移酶成员(UGTs)和小鼠的21个UGTs成员具有共同的尿苷二磷酸-葡萄糖醛酸和尿苷二磷酸-葡萄糖醛酸转移酶保守功能域;家蚕和小鼠的细胞色素P450酶(CYP450)家族成员也具有CYP450保守的结构域和功能域。家蚕和小鼠的药物代谢关键酶的结构与功能域上的保守性,提示两者在主要的解毒过程与作用机理中可能存在共同的特点。5.家蚕中肠是可建立肝毒性模型的经口给药“首关效应”器官根据APAP经口经药家蚕组织中原药APAP、中间产物NAPQI分布特点,催化药物代谢反应的关键酶(GST、GCL、TPX、UGT、CYP450等)的活性变化结果可知,中间产物经中肠后产量明显提高,高剂量APAP可造成中肠的吸收功能障碍。中肠组织中CYP450酶活变化显著,中肠组织中GST、GCL、TPX、UGT酶活本底显著高于脂肪体,且相应代谢酶活性变化较为显著。表明在经口给药途径中,家蚕中肠是药物代谢的“首关效应”器官。在经口给药途径中,家蚕的中肠与脂肪体相比而言,作为外源药物的“首关效应”器官,类比哺乳类解毒器官(肝脏)更为准确。家蚕中肠是更合适的研究哺乳类肝毒性药物毒性机理的靶器官(组织)模型。6. 结论家蚕已具备实验动物化的基本条件,能够建立系统化、标准化的实验动物饲养管理、环境控制操作规范;家蚕与小鼠对肝毒性模式药物APAP的药物代谢动力学具有一致的特征;家蚕催化解毒作用的关键性代谢酶活性变化及作用机制与哺乳类相似。以家蚕为实验动物,建立中肠损伤模型,可以作为哺乳动物肝损伤模型的替代,用于研究哺乳动物的解毒机制和肝损机理。
[Abstract]:The alternative method of experimental animals has been widely used in the field of life science. The silkworm is a Lepidoptera model insect, which has thousands of years of animal husbandry history, and has accumulated systematic and deep physiological, pathological and toxicological data. The innovation of the genetic mutation resources of the silkworm and its basic life system, material metabolism, energy metabolism and heredity. There are many similarities between mammals and mammals, which make the silkworm as the Chinese characteristic model insects are gradually developing to the model of experimental animals. However, how to realize the "experimental animals" of silkworm by systematic control of the genetic factors and the growth environment of the silkworm, how to choose the alternative according to the biological characteristics of the silkworm. The study type is the bottleneck that the silkworm must break through in the process of becoming a standard experimental animal. In addition, what are the similarities and differences in the metabolic kinetics of foreign compounds compared with the classic mammalian animal models, such as silkworm and mice, and there are no systematic studies at home and abroad. For the purpose of construction, a standardized breeding micro barrier system for silkworm, silkworm, the standardized feeding system of silkworm, was designed, and the acute toxicity classification and pharmacokinetics of APAP in silkworm and mammalian moving objects were compared and studied by the hepatotoxicity model drug (APAP). By means of metabonomics and high throughput analysis (RNA-Seq), the difference of endogenous metabolites and gene expressions of APAP in silkworm was investigated, and the biological effects of APAP in the body were revealed. The activity of the key enzyme in the catalytic drug metabolism of the silkworm in the Bombyx mori after APAP was detected by the kit, and the silkworm and mice were compared by bioinformatics. The difference in the evolution of these key enzymes and the difference in the active site of these key enzymes. The main results are as follows: the acute toxicity classification of 1. model drug APAP to the silkworm, the pharmacokinetic characteristics can be replaced by the acute toxicity test of the parallel data for the replacement of the medical experimental animals, and the toxicity of APAP to the silkworm is different. The sensitivity was different, and the.APAP treatment was more sensitive than that of the white moon. The LD50 of the male species was 2017 + 254 g/g and 2169 + 300 g/g. respectively. The acute toxicity of the male was similar to the mammalian. The metabolic kinetics showed that the pharmacokinetics of the low concentration 600 mu g/g and the high concentration of 3600 micron g/g in the silkworm was open in one room. The elimination half life (t1/2) was 1.06h and 2.77h, respectively, and the peak time (Tmax) of the original drug was 0.50h and 1.00h respectively; the peak concentration (Cmax) of the original drug was 67.69 mu g/ml and 568.73 micron relative bioavailability (AUC0-t) respectively: 222.63 h, micron and 1976.32? Compared with milk, the drug absorption distribution, metabolic intermediate products in the body, and the overall dynamics (PK) are similar. It can replace the parallel data.2. model drug APAP for the medical experimental animals to produce the common biological effect metabolomics analysis in the silkworm in the silkworm, and the difference generation in the circulating blood after the 8 h of the Bombyx Mori was given to the Bombyx mori with the dose of 600 mu g /g. The metabolites in the three carboxylic acid cycle, glycolysis pathway, amino acid metabolism, melanogenesis, etc., indicate that the toxic pathways produced by APAP in the body are mainly caused by oxidative stress during the metabolic process, affecting the energy supply, transport and signal transduction in the body. The analysis of metabolite differences found that APAP induces ammonia in the haemolymph of silkworm. Differences in basal acid metabolism suggest that abnormality in amino acid metabolism is associated with a significant decline in endogenous metabolites such as malic acid, succinic acid and Corydalis, and also associated with oxidative stress produced by APAP. A significant increase in tyrosine, DOPA and trehalose further confirms that excessive APAP leads to oxidative stress; APAP induces silkworm gall. The decrease of sterol and the increase of beta hydroxy beta methylglutaric acid indicates that APAP leads to the imbalance of cholesterol synthesis pathway in silkworm, steroid hormone supply barrier affects the hormone regulation of the body's hormone regulation.3. model drug APAP can produce the common pharmacological action and toxic mechanism of mammalian in the silkworm in the body of the silkworm, RNA-Seq results analysis of the APAP administration experiment group and the pair The difference gene expression in the group showed that in the KEGG analysis system, a large number of differentially expressed genes were enriched in the three carboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, glycolysis and sugar isogenesis, and further demonstrated that the damage to silkworm organism by APAP is mainly oxygen from the gene expression level. In the KEGG analysis system associated with the organism system, a large number of differentially expressed genes are enriched in cardiac muscle contraction, vascular smooth muscle contraction, and actin framework regulation. Related pathways and partial neuronal synaptic gene pathways, which are consistent with the results of metabolomics. It is speculated that APAP has the pharmacological action of inhibiting the four enoic acid pathway of the silkworm, which is consistent with mammalian,.4., the key enzyme of the drug metabolism of the silkworm and mice, has the common active site and protein interaction mechanism bioinformatics analysis results. The family members of the glutathione transferase family (GSTs) in the silkworm and mouse showed that most of the domain and functional domain had the GSTs active region and the folding pattern of thioredoxin; the glutamate cysteine ligase catalyzed subunit (GCLc) protein of the silkworm and mice all had the functional domain of the glutathione synthetase, and the glutamate cysteine linkage The enzyme (GCLM) has NADP dependent oxidoreductase function domain; the peroxidase protein encoded by the Jafrac1 gene in the silkworm thioredoxin peroxidase family (TPXs) has a highly conserved structure with the peroxidase encoded by the Prdx1 and Prdx2 genes in mice, and the predicted protein LOC733003, LOC101735759, and LOC732921 of the silkworm are respectively of the silkworm. The conservative domain of Prdx3, Prdx4, Prdx5 encoded proteins in mice; 2 members of glucuronotransferase (UGTs) in silkworm and 21 UGTs members of mice have the same conservative domain of uridine two phosphoric acid glucuronic acid and uridine two phosphate glucuronide transferase; the family of cytochrome P450 enzyme (CYP450) family in silkworm and mice is also a member of the family. CYP450 conserved domain and functional domain. The structure and conservatism of the key enzymes in the drug metabolism of the silkworm and mice, suggesting that both of them may have the common characteristics in the main detoxification process and the mechanism of action.5. the middle intestine of the silkworm, silkworm, is the "first effect" organ of the oral administration of the liver toxicity model based on the mouth of the APAP The distribution of central product APAP, intermediate product NAPQI, the activity changes of the key enzymes (GST, GCL, TPX, UGT, CYP450, etc.) in the drug metabolism reaction of the Bombyx mori tissue, the output of the intermediate products obviously increased after the midgut, and the high dose of APAP could cause the absorption dysfunction of the midgut. The change of the CYP450 enzyme activity in the midgut tissue was significant and the middle intestinal tissue was in the middle intestine. The GST, GCL, TPX, UGT enzyme activity was significantly higher than that of the fat body, and the activity of the corresponding metabolic enzyme changed significantly. It showed that in the oral administration route, the middle intestine of the silkworm was the "first effect" organ of the drug metabolism. Mammalian detoxification organ (liver) is more accurate. Silkworm midgut is a more suitable target organ (tissue) model to study the toxicity mechanism of mammalian toxic drugs to the liver.6. conclusion silkworm has the basic conditions for experimental animals. It can establish systematized, standardized experimental animal feeding management, environmental control operation norms, silkworm and mice to liver. The metabolic kinetics of the toxic model drug APAP has the same characteristics. The changes in the activity of the key metabolic enzymes and the mechanism of action of the silkworm are similar to those of mammalian. The model of midgut injury is established by the silkworm as the experimental animal, which can be used as a substitute for the model of mammalian liver injury and is used to study the detoxification mechanism of mammals. And the mechanism of liver damage.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332
本文编号：2125778
[Abstract]:The alternative method of experimental animals has been widely used in the field of life science. The silkworm is a Lepidoptera model insect, which has thousands of years of animal husbandry history, and has accumulated systematic and deep physiological, pathological and toxicological data. The innovation of the genetic mutation resources of the silkworm and its basic life system, material metabolism, energy metabolism and heredity. There are many similarities between mammals and mammals, which make the silkworm as the Chinese characteristic model insects are gradually developing to the model of experimental animals. However, how to realize the "experimental animals" of silkworm by systematic control of the genetic factors and the growth environment of the silkworm, how to choose the alternative according to the biological characteristics of the silkworm. The study type is the bottleneck that the silkworm must break through in the process of becoming a standard experimental animal. In addition, what are the similarities and differences in the metabolic kinetics of foreign compounds compared with the classic mammalian animal models, such as silkworm and mice, and there are no systematic studies at home and abroad. For the purpose of construction, a standardized breeding micro barrier system for silkworm, silkworm, the standardized feeding system of silkworm, was designed, and the acute toxicity classification and pharmacokinetics of APAP in silkworm and mammalian moving objects were compared and studied by the hepatotoxicity model drug (APAP). By means of metabonomics and high throughput analysis (RNA-Seq), the difference of endogenous metabolites and gene expressions of APAP in silkworm was investigated, and the biological effects of APAP in the body were revealed. The activity of the key enzyme in the catalytic drug metabolism of the silkworm in the Bombyx mori after APAP was detected by the kit, and the silkworm and mice were compared by bioinformatics. The difference in the evolution of these key enzymes and the difference in the active site of these key enzymes. The main results are as follows: the acute toxicity classification of 1. model drug APAP to the silkworm, the pharmacokinetic characteristics can be replaced by the acute toxicity test of the parallel data for the replacement of the medical experimental animals, and the toxicity of APAP to the silkworm is different. The sensitivity was different, and the.APAP treatment was more sensitive than that of the white moon. The LD50 of the male species was 2017 + 254 g/g and 2169 + 300 g/g. respectively. The acute toxicity of the male was similar to the mammalian. The metabolic kinetics showed that the pharmacokinetics of the low concentration 600 mu g/g and the high concentration of 3600 micron g/g in the silkworm was open in one room. The elimination half life (t1/2) was 1.06h and 2.77h, respectively, and the peak time (Tmax) of the original drug was 0.50h and 1.00h respectively; the peak concentration (Cmax) of the original drug was 67.69 mu g/ml and 568.73 micron relative bioavailability (AUC0-t) respectively: 222.63 h, micron and 1976.32? Compared with milk, the drug absorption distribution, metabolic intermediate products in the body, and the overall dynamics (PK) are similar. It can replace the parallel data.2. model drug APAP for the medical experimental animals to produce the common biological effect metabolomics analysis in the silkworm in the silkworm, and the difference generation in the circulating blood after the 8 h of the Bombyx Mori was given to the Bombyx mori with the dose of 600 mu g /g. The metabolites in the three carboxylic acid cycle, glycolysis pathway, amino acid metabolism, melanogenesis, etc., indicate that the toxic pathways produced by APAP in the body are mainly caused by oxidative stress during the metabolic process, affecting the energy supply, transport and signal transduction in the body. The analysis of metabolite differences found that APAP induces ammonia in the haemolymph of silkworm. Differences in basal acid metabolism suggest that abnormality in amino acid metabolism is associated with a significant decline in endogenous metabolites such as malic acid, succinic acid and Corydalis, and also associated with oxidative stress produced by APAP. A significant increase in tyrosine, DOPA and trehalose further confirms that excessive APAP leads to oxidative stress; APAP induces silkworm gall. The decrease of sterol and the increase of beta hydroxy beta methylglutaric acid indicates that APAP leads to the imbalance of cholesterol synthesis pathway in silkworm, steroid hormone supply barrier affects the hormone regulation of the body's hormone regulation.3. model drug APAP can produce the common pharmacological action and toxic mechanism of mammalian in the silkworm in the body of the silkworm, RNA-Seq results analysis of the APAP administration experiment group and the pair The difference gene expression in the group showed that in the KEGG analysis system, a large number of differentially expressed genes were enriched in the three carboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, glycolysis and sugar isogenesis, and further demonstrated that the damage to silkworm organism by APAP is mainly oxygen from the gene expression level. In the KEGG analysis system associated with the organism system, a large number of differentially expressed genes are enriched in cardiac muscle contraction, vascular smooth muscle contraction, and actin framework regulation. Related pathways and partial neuronal synaptic gene pathways, which are consistent with the results of metabolomics. It is speculated that APAP has the pharmacological action of inhibiting the four enoic acid pathway of the silkworm, which is consistent with mammalian,.4., the key enzyme of the drug metabolism of the silkworm and mice, has the common active site and protein interaction mechanism bioinformatics analysis results. The family members of the glutathione transferase family (GSTs) in the silkworm and mouse showed that most of the domain and functional domain had the GSTs active region and the folding pattern of thioredoxin; the glutamate cysteine ligase catalyzed subunit (GCLc) protein of the silkworm and mice all had the functional domain of the glutathione synthetase, and the glutamate cysteine linkage The enzyme (GCLM) has NADP dependent oxidoreductase function domain; the peroxidase protein encoded by the Jafrac1 gene in the silkworm thioredoxin peroxidase family (TPXs) has a highly conserved structure with the peroxidase encoded by the Prdx1 and Prdx2 genes in mice, and the predicted protein LOC733003, LOC101735759, and LOC732921 of the silkworm are respectively of the silkworm. The conservative domain of Prdx3, Prdx4, Prdx5 encoded proteins in mice; 2 members of glucuronotransferase (UGTs) in silkworm and 21 UGTs members of mice have the same conservative domain of uridine two phosphoric acid glucuronic acid and uridine two phosphate glucuronide transferase; the family of cytochrome P450 enzyme (CYP450) family in silkworm and mice is also a member of the family. CYP450 conserved domain and functional domain. The structure and conservatism of the key enzymes in the drug metabolism of the silkworm and mice, suggesting that both of them may have the common characteristics in the main detoxification process and the mechanism of action.5. the middle intestine of the silkworm, silkworm, is the "first effect" organ of the oral administration of the liver toxicity model based on the mouth of the APAP The distribution of central product APAP, intermediate product NAPQI, the activity changes of the key enzymes (GST, GCL, TPX, UGT, CYP450, etc.) in the drug metabolism reaction of the Bombyx mori tissue, the output of the intermediate products obviously increased after the midgut, and the high dose of APAP could cause the absorption dysfunction of the midgut. The change of the CYP450 enzyme activity in the midgut tissue was significant and the middle intestinal tissue was in the middle intestine. The GST, GCL, TPX, UGT enzyme activity was significantly higher than that of the fat body, and the activity of the corresponding metabolic enzyme changed significantly. It showed that in the oral administration route, the middle intestine of the silkworm was the "first effect" organ of the drug metabolism. Mammalian detoxification organ (liver) is more accurate. Silkworm midgut is a more suitable target organ (tissue) model to study the toxicity mechanism of mammalian toxic drugs to the liver.6. conclusion silkworm has the basic conditions for experimental animals. It can establish systematized, standardized experimental animal feeding management, environmental control operation norms, silkworm and mice to liver. The metabolic kinetics of the toxic model drug APAP has the same characteristics. The changes in the activity of the key metabolic enzymes and the mechanism of action of the silkworm are similar to those of mammalian. The model of midgut injury is established by the silkworm as the experimental animal, which can be used as a substitute for the model of mammalian liver injury and is used to study the detoxification mechanism of mammals. And the mechanism of liver damage.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332
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