利用斑马鱼胚胎评价秋水仙碱的毒性及其机制研究

发布时间：2018-06-10 01:19

本文选题：秋水仙碱 + 斑马鱼幼鱼　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的：1.维持斑马鱼模式集中养殖与繁育系统稳定运行，为实验开展提供优质胚胎、幼鱼和成鱼。2.整体水平上探讨秋水仙碱对斑马鱼幼鱼体内SOD、Na+-K+-ATPase活性和MDA、GSH含量的影响3.观察秋水仙碱对斑马鱼幼鱼体内抗氧化酶表达相关基因bcl2、gstp2、ucp2、nqo1、sod1的表达情况。4.局部水平上研究秋水仙碱对斑马鱼幼鱼体内肝脏Shh、Gli3蛋白表达以及肝脏脂肪含量的影响。方法：1.在斑马鱼模式生物集中繁育系统稳定运行基础上，获取胚胎，培育幼鱼和成鱼用于实验。2.药物暴露实验按如下方法进行：预实验基础上确定幼鱼药物暴露72h最大不致死浓度（MNLC），将3dpf斑马鱼幼鱼随机分为五组，给药浓度分别为0.2MNLC、0.5MNLC、1.0MNLC、LC10，以Holt Buffer为空白对照组，药物作用72h，进行各项指标测定：（1）抗氧化酶活性及抗氧化物含量测定。将幼鱼充分研磨，制成组织匀浆，离心，取上清，测定幼鱼体内超氧化物歧化酶（SOD）、钠-钾-三磷酸腺苷酶（Na+-K+-ATPase）活性和谷胱甘肽（GSH）、丙二醛（MDA）含量。（2）抗氧化酶相关基因表达情况。利用RT-PCR方法测定药物作用72h后斑马鱼幼鱼体内抗氧化酶表达相关基因bcl2、gstp2、ucp2、nqo1、sod1的表达情况。（3）肝组织病理变化以及Shh、Gli3蛋白表达情况测定。4%多聚甲醛磷酸液4℃固定24h，脱水，石蜡包埋，切片，对斑马鱼幼鱼进行HE染色、油红染色，并采用免疫组化方法分析斑马鱼幼鱼体内Shh、Gli3蛋白表达变化情况。结果：1.斑马鱼模式生物集中繁育体系能够为实验开展提供优质胚胎，繁育幼鱼和成鱼用于实验。2.秋水仙碱对6dpf斑马鱼幼鱼的MNLC为20.6063μg mL-1，LC10为26.0615μg mL-1，LC50为34.7536μg mL-1。与空白对照组相比，随着秋水仙碱浓度增大，斑马鱼幼鱼SOD和Na+-K+-ATPase活性均降低；MDA含量随着浓度增大而升高；GSH含量随着浓度增大，含量降低。3.与空白对照组相比，随着秋水仙碱浓度增加，各给药组bcl2-mRNA、gstp2-mRNA、nqo1-mRNA、sod1-mRNA表达量减少。而ucp2-mRNA表达量与对照组相比则呈上升趋势。4.与空白对照组比较，随着秋水仙碱浓度增加，HE染色结果显示，，高剂量组的幼鱼肝组织变性，空泡增多，细胞间距增加。油红染色结果显示斑马鱼肝脏颜色加深，提示肝脏中脂肪含量增加；免疫组化法测定斑马鱼肝脏中Shh、Gli3蛋白表达量，随着药物浓度增加，两种蛋白含量表达增加，提示秋水仙碱对肝脏损伤作用可能跟激活Hedgehog信号通路上相关成分有关。结论：1.斑马鱼模式生物集中繁育体系稳定运行，可为药物安全性评价提供胚胎、幼鱼、成鱼三种时期的实验对象，扩大了应用斑马鱼进行药物安全性评价的应用范围。2.以斑马鱼幼鱼为实验对象，研究秋水仙碱对斑马鱼抗氧化系统的影响，表现为SOD和Na+-K+-ATPase活性受到抑制，GSH含量降低，MDA含量显著增加，导致幼鱼体内氧化还原状态失调。秋水仙碱对斑马鱼幼鱼毒性作用可能与此相关。秋水仙碱对斑马鱼幼鱼肝脏毒性的机制可能与激活Hedgehog信号通路有关。
[Abstract]:Objective: 1. to maintain the stable operation of the centralized aquaculture and breeding system of zebrafish, to provide high quality embryos, young fish and adult.2. on the overall level of the fish, the effect of colchicine on SOD, Na+-K+-ATPase activity and MDA, GSH content in zebrafish young fish 3. to observe the expression of colchine on the expression of antioxidant enzymes in zebrafish young fish Expression of gene BCL2, gstp2, UCP2, NQO1, SOD1, the effect of colchicine on the liver Shh, Gli3 protein expression and liver fat content in the liver of zebrafish young fish was studied at.4. level.
Methods: 1. on the basis of the stable operation of the zebrafish model biological centralized breeding system, obtaining embryos, cultivating young fish and adult fish for experimental.2. drug exposure experiments were carried out as follows: the maximum unlethal concentration of 72h (MNLC) was determined on the basis of pre experiment, and the young fish of 3dpf zebrafish were randomly divided into five groups, and the concentration of drug delivery was respectively 0.2MNLC, 0.5MNLC, 1.0MNLC, LC10, Holt Buffer as the blank control group, the drug action 72h, and the determination of various indexes: (1) antioxidant enzyme activity and antioxidant content determination. Young fish are fully lapping, tissue homogenate, centrifugation, Torikami Kiyo, determination of infant body superoxide dismutase (SOD), sodium potassium ATPase (Na+-K+-ATPas) E) activity and glutathione (GSH), malondialdehyde (MDA) content. (2) expression of antioxidant enzymes related genes. The expression of antioxidant enzymes related genes BCL2, gstp2, UCP2, NQO1, SOD1 in juvenile zebrafish after 72h was determined by RT-PCR method. (3) pathological changes of liver tissue and Shh, Gli3 protein expression determination of polymethylene Aldehyde phosphate solution was fixed at 4 C for 24h, dehydrated, paraffin embedded and sliced to stain the young zebrafish fish by HE staining and oil red staining. The changes of Shh and Gli3 protein expression in the young fish of zebrafish were analyzed by immunohistochemical method.
Results: 1. zebrafish model biological concentration breeding system can provide high quality embryos for experimental development, breeding young fish and adult fish for experimental.2. colchicine to 6dpf zebrafish young fish MNLC 20.6063 g mL-1, LC10 26.0615 mu g mL-1, LC50 34.7536 mu g mL-1. compared with the blank control group, with colchicine concentration increases, zebra fish The activity of SOD and Na+-K+-ATPase decreased in young fish, and the content of MDA increased with the concentration, and the content of GSH increased with the concentration, and the content of.3. decreased as the concentration of colchicine increased, with the increase of colchicine concentration, the expression of bcl2-mRNA, gstp2-mRNA, nqo1-mRNA, and sod1-mRNA decreased as the concentration of colchicine increased, while the expression of ucp2-mRNA was higher than that of the control group. Compared with the blank control group, with the increase of colchicine concentration, the results of HE staining showed that the liver tissue denatured, the vacuoles increased and the cell spacing increased in the high dose group. The oil red staining results showed that the liver color of zebrafish was deepened and the fat content in the liver was increased, and the immunohistochemical method was used to determine Shh, Gli3 protein in the liver of zebrafish. The expression of the two proteins increased as the concentration of the drug increased, suggesting that colchicine may be associated with the activation of the related components on the Hedgehog signaling pathway.
Conclusion: 1. zebrafish model biological centralized breeding system is stable operation, which can provide the experimental object of three periods of embryo, young fish and adult fish for the evaluation of drug safety. The application scope of the drug safety evaluation of zebrafish with zebrafish was expanded by.2., and the effects of colchicine on the anti oxidation system of zebrafish were studied. The activity of SOD and Na+-K+-ATPase was inhibited, the content of GSH decreased and the content of MDA increased significantly, which resulted in the maladjustment of redox state in the young fish. The toxicity of colchicine to zebrafish young fish may be related to this. The mechanism of colchicine's toxicity to the liver of zebrafish can be related to the activation of the Hedgehog signaling pathway.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【参考文献】