使用秀丽隐杆线虫作为模型测定小分子药物的驱肠虫活性

Viviane Sant’Anna; Wanderley de Souza; Rossiane  C. Vommaro

doi:10.21769/BioProtoc.2113

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Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model

使用秀丽隐杆线虫作为模型测定小分子药物的驱肠虫活性

Viviane Sant’Anna

Wanderley de Souza

Rossiane C. Vommaro email

发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2113 浏览次数: 11339

评审: Neelanjan BoseAnonymous reviewer(s)

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Cover of Experimental Parasitology, featuring study using the protocol.

Apr 2016

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Abstract

This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in embryonated eggs, larval stages and in the adults’ survival. As most current anthelmintics are not effective against all nematode life stages, this protocol can contribute to the identification of new alternatives to helminthic infections (Sant’Anna et al., 2016).

Keywords: C. elegans (秀丽隐杆线虫)

Nematodes (线虫)

Anthelmintic drugs (驱虫药)

Chemotherapy (化学疗法)

Background

Caenorhabditis elegans is a model organism for parasite nematode research and an excellent system for the screening of compounds with potential anthelmintic activity, because it is inexpensive, readily available, and easy to work with. In addition, the use of C. elegans in assays to investigate nematode behavior, locomotion, reproduction and death is uncomplicated and reliable (Simpkin and Coles, 1981). The protocols for screening new compounds on C. elegans were first carried out in axenic liquid medium in deep well microscope slides (Tomlinson et al., 1985) or using the drugs added to melted modified NGM agar (Driscoll et al., 1989). These methods are not effective in drug screening as axenic cultures, containing low food supply, trigger the intra-uterine birth causing maternal death (endotokia matricida) (Lenaerts et al., 2008) and drugs added to melted agar can modify drug stability due to the high temperatures. In this protocol, we used 96-well plates with liquid medium supplied with Escherichia coli to evaluate each stage (eggs, L1-L2 larvae, L3-L4 larvae and adults) independently.

Materials and Reagents

Transfer pipette
15 ml Falcon tubes (Corning, Falcon^®, catalog number: 352095 )
96-well plate, flat bottom, polystyrene, 0.32 cm², sterile. TPP tissue culture plates (Sigma-Aldrich, catalog number: Z707910 )
Tissue culture dishes of polystyrene TPP- diam. 60 x 15 mm, surface area size 22.1 cm² with NGM (Sigma-Aldrich, catalog number: Z707678 )
C. elegans N2 strain
Escherichia coli OP50 strain
Drugs to screen
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 795429 )
Hypochlorite (NaClO) (Sigma-Aldrich, catalog number: 13440 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S5136 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O), BioUltra ≥ 99.5% (KT) (Sigma-Aldrich, catalog number: 63138 )
Potassium phosphate dibasic (K₂HPO₄), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: P3786 )
Cholesterol (Sigma-Aldrich, catalog number: C3045 )
Ethanol (p.a., without additive, ≥ 99.8%) (Sigma-Aldrich, catalog number: 24102 )
Note: This product has been discontinued.
Citric acid monohydrate (ACS reagent, ≥ 99.0%) (Sigma-Aldrich, catalog number: C1909 )
Tri-potassium citrate monohydrate (Sigma-Aldrich, catalog number: 6100-05-6 )
Disodium EDTA (98.5-101.5%, BioUltra) (Sigma-Aldrich, catalog number: E1644 )
Iron (II) sulfate heptahydrate (FeSO₄·7H₂O) (Sigma-Aldrich, catalog number: 215422 )
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sigma-Aldrich, catalog number: 203734 )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (BioReagent, suitable for cell culture) (Sigma-Aldrich, catalog number: 7446-20-0 )
Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (BioReagent, suitable for cell culture, ≥ 98%) (Sigma-Aldrich, catalog number: C8027 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C1016 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
Lysing solution (see Recipes)
M9 buffer (1 L) (see Recipes)
S medium (1 L) (see Recipes)

Equipment

Clinical centrifuge (Thermo Fisher Scientific, catalog number: 22-029-416 )
Inverted microscope (ZEISS, model: Axio Vert.A1 )
Biochemical oxygen demand (BOD) incubator (Thermo Fisher Scientific, Fisher Scientific^TM, catalog number: 37-20 )
Micropipet, 100-1,000 μl volume (Nichipet Eco pipette, catalog number: Z710199 )
Autoclave

Procedure

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Sant’Anna, V., de Souza, W. and Vommaro, R. C. (2017). Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model. Bio-protocol 7(2): e2113. DOI: 10.21769/BioProtoc.2113.