全氟辛烷磺酸对斑马鱼脂代谢和糖代谢毒理作用研究-遗传学专业毕业论文.pdf温州医学院硕士学位论文全氟辛烷磺酸对斑马鱼脂代谢和糖代谢毒理作用研究摘要全氟辛烷磺酸(Perfluorooctanesulfonic acid，PFOS)是一种应用广泛的全氟化表面活性剂。
现有的研究表明：PFOS已经广泛渗透到地球的各种环境介质中，甚至包括地球的两极；是一类具有全身多脏器毒性的环境污染物：包括遗传毒性、雄性生殖毒性、神经毒性、发育毒性和内分泌干扰作用等。
但是目前还鲜见其在糖脂代谢上的研究，在水生生物中的研究和数据更是匮乏。
我们以野生型AB系斑马鱼为研究对象，研究在低剂量、长时间暴毒下，PFOS是否以及如何影响脂类、糖类代谢。
实验可分为两个部分：成鱼实验：亲代斑马鱼，自8hpf开始暴露于一系列不同浓度(0，0．02，0．1，0．5I．tM)的PFOS中。
暴露150天，固定其中的一部分，用于制备组织病理切片，以观察其内脏组织结构；固定另一部分内脏，用于制备透射电镜超微切片，研究PFOS暴露对肝细胞和小肠上皮细胞超微结构的影响。
暴露180天，从各PFOS暴露组分别选取成熟的雌雄斑马鱼繁殖F1代，并且分为持续暴毒组和清水脱毒组继续养殖至50dpf，分别制备病理切片观察组织结构；检测暴露180天后，亲代斑马鱼的各项血液生化指标，包括血糖，血清甘油三酯，血清总胆固醇；同时检测斑马鱼的基本生长指数：肥满度和肝脏指数。
．仔鱼实验：运用荧光定量PCR技术，分别检测96hpf急性窗口期暴毒斑马鱼样本代谢相关基因的表达变化和F1代脱毒斑马鱼样本相关基因的表达变化。
利用IPA构建两个信号通路，分析基因间的调控关系。
研究发现了较为明显的由PFOS引起的各种体内糖脂代谢的紊乱现象：HE 染色的病理切片表明，暴露时间150天的亲代0．51xM组雄鱼普遍存在肝脏脂肪化积累现象；由亲代0．1斗M PFOS暴露组所产脱毒50天养殖的F1代，雌鱼和雄鱼都出现了明显的肝脏脂肪累积，脂肪化程度比在亲代中稍低。
对暴露时间达180天的斑马鱼进行血糖、血清甘油三酯和总胆固醇的检测，表明血糖受影响不大，只在O．1“M组雄鱼有小幅上升；血清甘油三酯，随着PFOS暴露浓度的升高有显著降低的趋势；血清总胆固醇，在雌鱼和雄鱼中检测的结果有明显差异。
对同时期鱼进行生长指数的测定结果，肥满度在0．51xM组显著升高和肝体指数在各暴露组显著降低，表明PFOS对斑马鱼基本代谢状况确实存在影响。
对暴露150天的斑马鱼肝脏和小肠进行透射电镜超微切片，发现了脂肪化程度严重的肝细胞(雄鱼)：在超微结构上，细胞内可见大大小小的脂滴，肝糖原降低，且部分细胞核因脂质的挤压而发生变形；细胞内胆小管的微绒毛变稀疏、排列紊乱；细胞间胆小管数量下降；线粒体嵴模糊；肝细胞超微结构中与物质运1温州医学院硕士学位论文输有关的细胞器受损伤，推测脂质运输的环节出了问题。
同组雄鱼的小肠上皮细胞的微绒毛根部出现部分脱落。
对成年斑马鱼的研究都按雌雄分组进行，结果也反映了PFOS在不同性别的鱼之间毒性有差异；大部分实验的结果支持雄鱼受PFOS的影响更大。
推测是雌鱼通过产卵途径释放出了部分体内的PFOS。
对96hpf仔鱼的一些基因表达水平的研究，发现脂代谢和糖代谢通路中的多个基因表达变化：比如，dgatlb，hb9，kissl在急性样本和F1代样本均有一致的显著变化：xdh在F1代的表达显著上升。
最后在筛选一些肥胖症相关基因中，发现leptin在Fo和F1代样本中的表达都发生了显著上升。
这些数据引导我们进一步深入研究PFOS干扰糖脂代谢的机制。
关键词：PFOS斑马鱼脂代谢F1代却砌2温州医学院硕士学位论文Altered Lipid Metabolism and Glucose Metabolism inZebrafish Exposed to Perfluorooctanesuiphonic AcidAbstractPerfluorooctanesulphonic acid(PFOS)has been used widely in commercial and industrial applications as a surfactant．Considerable r esearches h ave shown t hat PFOS iswidespread globally in many environmental media，including remote regions far fromsources，such as the Arctic and Antarctic．Meanwhile，it is an environmental pollutant with multiple toxicities including genotoxicity,reproductive toxicity, neurotoxicity,developmental toxicity and endocrine disruption effect．However，very little is known about the toxicity of PFOS on disturbing lipid metabolism or glucose metabolism，especially in aquatic ecosystems．In this research，we investigate the impact of long—term exposure to low concentrations of PFOS in wild-type AB line zebrafish，and try to figure out whether PFOS disturbs lipid or glucose homeostasis，and how it works．The research can be divided into two parts，as follows：Experiments upon adult fish：zebrafish fry(Fo，8 hours post fertilization，hpf)，were exposed to 0(contr01)，0．02，0．1 and 0．5“M PFOS until 150 days post fertilization(dpf)．At150 dpf,we fixed 8 fish of each PFOS and control groups for histological examination，and fixed the tissue of the liver and the intestine for transmissionelectron microscopy(TEM)．At 180 dpf,the F1 embryos were derived from parental PFOSexposure and control groups and then bred in clean water or continued exposure in the same PFOS dose until 50 dpf．And then the fish were treated in thesame way as above for histological examination．Fasting blood glucose level，serum triglycerides and sernm total cholesterol were assayed for parent groups exposed to 180dpf．Experiments upon larval fish(including F0 larve，F1 offspring)：expression levels of genes related to lipid metabolism and glucose metabolism were detected by real—timeAnalysis(IPA) quantification PeR(qPCR)．Then we used Ingenuity Pathwaysoftware to create lipid metabolism pathway and glucose metabolism pathway toanalyze those tested genes．Generally,we find numerous evidences to support that PFOS disturbed lipid or3温州医学院硕士学位论文glucose homeostasis．In FO generation，the most marked change in liver sections of male fish exposed to 0．5 pM PFOS was vacuolization，mainly due to accumulation of lipid droplets．The same but tender phenomenon was found in 50 dpf F1 offspring bred in clean water．There’S slightly change in fasting blood level，and serum triglycefides were significantly reduced，while cholesterol levels were some unusual．Condition factor and hepatosomatic index，especially the latter,was altered considerably．A transmission electron microscope was used to examine the ultrastructure of liver and small intestine in 150 dpf PFOS exposed adult fish．In liver,cells of 0．5¨M male fish we observed severe hepatocellular lipid droplets accumulation，while glycogen decreased significantly；intracellular canaliculi decreased and disordered，mitochondrial crista was hardly to distinguish．We concluded that organelles related substances transportation was damaged，which was important to lipid transportation in and bet’Ⅳeen cells．In experiments conduct．ed in the adult fish，the male and female were strictlyseparated，while corresponding results also reflected variances between fish+of different genders．A majority of results support that the male were more sensitive to PFOS．We may considerately confer that the female released PFOSby spawning．QPCR was performed on genes related to lipid metabolism and glucose metabolism；and several genes were altered in both FO larvae and F1 offspring．Take dgatlb，hb9，kissl for example，they a ll changed s ignificantly in both samples，while expression ofxdh increased only in F1 offspring．Finally,in the screening of some obesity-related genes，we found a significant increase in leptin expression in F0 larvae and F1 offspring．These data enlighten US to the further study concerning the mechanism of PFOSand glucose metabolism．disturbing lipidKeywords：zebrafish；PFOS；lipid metabolism；F1 generation；卸砌4温州医学院硕士学位论文————————————————————————————————————————————————————一一缩略词表缩略词英文全称中文全称PFOS Peffluorooctanesulphonic acid 全氟辛烷磺酸DMSODimethyl sulfoxide 二甲基亚砜EM Embryo mcdium 胚胎培养液hpf／dpf hours／days post fertilization 受精后几小时／天PBS Phosphate Buffer Solution 磷酸缓冲液PFA多聚甲醛paraformaldehyde烷基磺酸盐同位氨基MS．222 Tdcame Methane Sulfonate苯甲酸乙酯HSl Hepatosomatic index 肝体指数IPA Ingenuity Pathway Analysis 信号通路分析Quantitative reverse transcriptaseqRT-PCR 定量PCR分析PCREDTA-2Na EDTA Disodium Salt 乙二胺四乙酸二钠温州医学院硕士学位论文．▲-‘．-·LI刖吾长达半个世纪以上的、全球广范围的应用，外加PFOS自身化学结构决定的难降解性、生物累积性，以及超出现有研究之外的不可预测的世代遗传毒性：这些因素迫使有关环境保护组织、政府机构逐步采取对PFOS应用的限制性措施。