灭螺药氯代水杨胺对湖北钉螺的作用机理研究

发布时间：2018-07-09 17:02

本文选题：湖北钉螺 + WPN　；参考：《武汉大学》2016年博士论文

【摘要】：日本血吸虫病是广泛流行于我国的人兽共患寄生虫病。湖北钉螺(Oncomelania hupensis)是日本血吸虫(Schistosoma japonicum)唯一的的中间宿主,在血吸虫病的传播中起着十分重要的作用。控制钉螺是血吸虫病综合防治策略中的重要措施,其中化学药物灭螺具有高效性和使用方法简单的特点,仍是目前最常用的灭螺手段,在控制钉螺的密度方面发挥重要作用。氯硝柳胺(WPN)是目前常用的,并被世界卫生组织推荐用于现场灭螺的唯一药物,但其灭螺机理仍未完全阐明。新型灭螺药氯代水杨胺(lv dai shui yang an, LDS)是WPN的衍生物,经实验室和现场实验证实其与WPN具有相当的灭螺效果,且具有成本低、对水生生物毒性小等优点。本文在组织学、酶学以及转录组学水平比较研究钉螺在LDS作用后与WPN、水作用后的差异,以揭示LDS的杀螺机理与WPN的异同。通过浸泡法使用WPN、LDS和水对钉螺浸杀3h、6h、12h、24h后,在确定其杀螺效果的基础上,利用HE染色和常规透射电镜观察其组织学变化,结果显示,两种药物的作用均造成肌肉细胞内肌动蛋白纤维的紊乱,细胞核、线粒体、内质网等细胞器的损伤,其中LDS处理组中可以观察到肌肉细胞中更严重的细胞核损伤以及肝脏细胞更严重的空泡化及内质网断裂现象。这些损伤表明在药物处理后钉螺的运动能力以及肝脏和能量代谢均受到抑制。通过酶组织化学染色以及软体组织匀浆测酶活的方法对乳酸脱氢酶(lactate dehydrogenase, LDH)等13种酶在两种药物处理后的活性变化进行检测。在两种灭螺药的作用下,糖类代谢相关的酶类活性除了肌肉中的LDH均受到抑制,应激反应相关的酶类活性相对于对照组均有先上升后下降的趋势。糖类代谢相关酶类活性的变化表明在两种药物处理下钉螺的糖类代谢模式出现改变,引起能量供应不足以及乳酸的堆积；应激反应相关的酶类活性变化则提示在药物处理的早期钉螺具有较强的应激反应以及在药物处理的后期肝脏以及神经系统的严重损伤。大体来说WPN和LDS的作用效果是类似的,均造成了钉螺的组织结构、神经信号传导、能量代谢、应激水平以及肝脏功能等方面的损伤或变化,但LDS处理后的钉螺肝脏表现出更严重的损伤,应激水平和有氧呼吸相关的酶活性也受到更强的抑制。在上述研究的基础上,进一步利用转录组测序筛选出了WPN/LDS处理后差异表达的254个unigenes。GO和KEGG分析表明这些差异表达unigenes与细胞结构病变、神经信号传导以及能量代谢的抑制有关,这与酶活性变化的结果是相符的。然而,考虑到转录后调控和蛋白水平调控对酶活性的影响,对潜在的酶相关基因及调控模式的完整鉴定和注释在对LDS的灭螺机理的探索过程中非常必要的。这个转录组数据能够为湖北钉螺在分子水平上的深入研究提供帮助,促进湖北钉螺基因组的研究,其中与灭螺机制相关的数据可以给将来钉螺的控制提供巨大的帮助。
[Abstract]:Schistosomiasis (Oncomelania hupensis), the only intermediate host of Schistosoma japonicum (Schistosoma japonicum), plays a very important role in the transmission of schistosomiasis. Control of Oncomelania is an important measure in the comprehensive prevention and control strategy of blood fluke disease. It is still the most commonly used means of snail control to control the density of Oncomelania Snail, which is the most commonly used method to control the density of Oncomelania snails. WPN is currently used commonly, and is recommended by WHO for the only drug used in the field snail. However, the mechanism of the snail control is still not fully elucidated. LV Dai Shui Yang an (LDS) is a derivative of WPN. Through laboratory and field experiments, it has been proved that it has considerable snail effect with WPN, and has the advantages of low cost and low toxicity to aquatic organisms. In this paper, the differences of Oncomelania snails after LDS action with WPN and water after the action of LDS were compared in the histology, enzymology and transcriptional level. The mechanism of snail killing of LDS is different from that of WPN. After immersing the snail with WPN, LDS and water to kill 3h, 6h, 12h, 24h by immersion method, on the basis of determining its snail killing effect, the histological changes are observed by HE staining and conventional transmission electron microscopy. The results show that the effects of the two kinds of drugs all cause the disorder of actin fiber in the muscle cells, the nucleus, The damage of mitochondria, endoplasmic reticulum and other organelles, in which the more serious nuclear damage in the muscle cells and the more severe vacuolization and endoplasmic reticulum fracture in the liver cells were observed in the LDS treatment group. These damage showed that the movement ability of Oncomelania and the liver and energy metabolites were suppressed after the treatment. Chemical staining and soft tissue homogenate measure enzyme activity to detect the activity changes of 13 enzymes, such as lactate dehydrogenase (lactate dehydrogenase, LDH), and other drugs treated by two drugs. Under the action of two kinds of snail, the enzyme activity related to carbohydrate metabolism is inhibited in addition to the LDH in the muscle, and the activity related enzyme activity The changes in carbohydrate metabolism related enzymes showed that the carbohydrate metabolism patterns of Oncomelania snails were changed under the treatment of two kinds of drugs, which resulted in the lack of energy supply and the accumulation of lactic acid. The changes in the activity of enzymes related to stress reaction suggested that the early Oncomelania snails were stronger in the drug treatment. Stress response and severe damage to the liver and nervous system at later stage of drug treatment. In general, the effects of WPN and LDS are similar, causing damage or change in the structure, nerve signal, energy metabolism, stress level and liver function of Oncomelania oncomelania, but the liver of Oncomelania snails treated by LDS is shown. On the basis of the above study, 254 unigenes.GO and KEGG analyses of differential expression after WPN/LDS treatment showed that these differences expressed unigenes and cell structural changes, neural signal transduction and the more severe injury. The inhibition of energy metabolism is related to the results of changes in enzyme activity. However, considering the effect of post transcriptional regulation and protein level regulation on enzyme activity, the complete identification and annotation of potential enzyme related genes and regulation patterns are necessary in the exploration of the mechanism of LDS. The deep research on the molecular level of Oncomelania Snail in Hubei provides help to promote the study of the genome of Oncomelania Snail in Hubei. The data related to the mechanism of snail mechanism can provide great help for the control of Oncomelania snails in the future.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R184.38

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