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Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni

机译:基于Echinostoma caproni的新发育幼虫的体外药敏试验的开发

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摘要

Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays.; Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo(R) and Vybrant(R) were tested as an alternative assay read-out method.; The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60% larvae excysted. NEL remained viable in culture medium for 5--7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications.; NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.
机译:棘皮虫病是主要的食源性皮肤病二甲双歧酶,而卡氏棘皮虫(Echinostoma caproni)物种可作为发现杀真菌剂的有用模型。当前的体外药物敏感性测定使用成年大肠埃希氏线虫,将其与候选药物一起孵育并在显微镜下对72小时的生存力进行评分。这项研究的目的是研究使用卡普罗尼大肠埃希氏菌的新成虫幼虫(NEL)进行体外药物测试,与成虫实验相比,这种方法更快,更经济,更合乎道德。幼虫是通过收集蜗牛中的尾cer并使用胰蛋白酶-胆汁盐兴奋法触发它们的兴奋而获得的。进行了有关该化学转化过程的各种参数以及适当的NEL培养条件的研究,并对发现进行了评估。将NEL和成年蠕虫与吡喹酮,曲本苯二胺,阿苯达唑和奎宁一起孵育,并在孵育后72小时进行显微镜评估。另外,比色标记刃天青,CellTiter-Glo和Vybrant作为替代的测定读出方法进行了测试。化学激发法成功地以约20-60%的幼虫被囊泡的速率成功地诱导了头孢埃希氏菌的囊肿。 NEL在培养基中保持存活5--7天。使用NEL进行体外药物分析的结果与使用与相同药物孵育的成年蠕虫的分析结果相吻合。没有任何标记物能够可靠地产生与NEL生存力或细胞毒性成比例的信号,而没有明显的并发症。 NEL足以进行体外药物测试。在进一步提高激发子产率和测定装置的实用性方面仍然存在挑战。解决这些问题也可以使用比色标记物改善读数。使用NEL符合实验室动物道德使用的3 R规则,并且可以大大提高体外针对这种肠道吸虫筛选药物的速率和可负担性。

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