Name: measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans
Uploaded: 2017-10-05T15:00:00
Description: Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

Denne undersøgelse blev støttet af Korea Institut for videnskab og teknologi murene forskning tilskud (2E27513) og den høje Value Added mad teknologi udvikling Program (IPET) finansieret af Ministeriet for landbrug, fødevarer og landdistriktspørgsmål (315067-03).

Bemærk: hele forsøget skal udføres i en ren isolerede laboratorium ved 20 ° C med lav støv og minimering af forurening under orm og bakteriel håndtering. Til dette formål, forsøg skal udføres under flammen af et alkohol lampe eller ved hjælp af en ren bænk.
 1. vedligeholdelse af C. elegans og æg forberedelse til den kemiske Test <ol><li>Bevar C. elegans N2 (var. Bristol) på en NGM agar plade fodret med live E. coli OP50 ved 20 ° C ...

<ol>
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Plant Sci. 7, 1667 (2016).</li><li>Kang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+topoisomerase+inhibitor,+daurinol,+suppresses+growth+of+HCT116+cells+with+low+hematological+toxicity+compared+to+etoposide.">A novel topoisomerase inhibitor, daurinol, suppresses growth of HCT116 cells with low hematological toxicity compared to etoposide.</a> Neoplasia. 13 (11), 1043-1057 (2011).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J. Vis. Exp. (47), e2293 (2011).</li><li>Zarse, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin/IGF1+signaling+extends+life+span+by+promoting+mitochondrial+L-proline+catabolism+to+induce+a+transient+ROS+signal.">Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal.</a> Cell Metab. 15 (4), 451-465 (2012).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+on+solid+media.">Measuring Caenorhabditis elegans life span on solid media.</a> J. Vis. Exp. 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I denne artikel vil beskrive vi toksicitet evaluering af kemikalier i C. elegans, en jord nematode, ved hjælp af etoposide som et giftstof eksempel. Til dette formål brugte vi to forsøgsbetingelser. I det første sæt, C. elegans blev dyrket på etoposide indeholdende plader fra æg til den unge voksne fase, og derefter orm fik lov til at lægge æg på normale NGM plader uden kemikalier. I det andet eksperimentelle sæt, blev C. elegans løbende behandlet med etoposide gennem hele forsøgsp...

Toksikologisk evaluering er afgørende for udviklingen af lægemidler, nutraceuticals, og cosmeceuticals samt risikovurdering af forskellige miljømæssige toksiner. Den gnavere model er en af de mest populære i vivo eksperimentelle systemer for denne toksikologi undersøgelse; Alternativt, ikke-pattedyr organismer såsom C. elegans er også almindeligt anvendt. Non-pattedyr toksikologisk evalueringsmodeller er fordelagtig på grund af ikke kun dyrenes etiske spørgsmål, men også deres bekvemmelighed og nytte i betragtning af omkostningseffektivitet, vedligeholde, hastighed og reproducerbarhed1,2 ,3,4.
C. elegans, en jord runde orm, er blevet udnyttet som en model dyr i forskellige grundlæggende og anvendt biologi og kemi forskning. Det er en 1 mm lange, gennemsigtig nematode, som blot vedligeholdes i fast eller flydende Nematode vækst medier (NGM) fodret med den bakterielle stamme Escherichia coli OP50. C. elegans har en kort levetid, og wild-type N2 C. elegans lægger ca 300 æg. Det er derfor let formeret skal anvendes som eksperimentelle materialer3,4,5. C. elegans har også været meget anvendt i de toksikologiske undersøgelser af mange stoffer og miljøgifte6,7,8,9.
Fordi mange anticancermedicin målrette hastigt dividere kræftceller, kan de også skade hurtigt dividere normale celler såsom knoglemarv, intestinale epitel og hårsækken celler. For eksempel målrette topoisomerase hæmmende anticancermedicin DNA replikation processen med kræftceller; Derfor, de også hæmme hurtigt dividere normale celler. Alle levende organisme har topoisomerases, og disse topoisomerase-hæmmere mest sandsynlige indvirkning miljømæssige økosystemer6,10,11. Således, en drug toksikologisk evaluering platform ved hjælp af en model dyr er værdifulde for både udviklingen af lægemidler og miljørisikovurderingen.
I denne artikel vil beskrive vi de detaljerede protokoller for at teste giftigheden af etoposide, som er en klinisk anticancer agent at mål topoisomerase II, som en model giftige kemikalie i C. elegans. Til dette formål, vil vi beskrive målemetode kropsstørrelse og det samlede antal æg lagt i C. elegans behandlet med etoposide.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Affymetrix, USA</td>
			<td>10906</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma, USA</td>
			<td>C3045</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma, USA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma, USA</td>
			<td>E1383</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software (ver 1.4)</td>
			<td>Natinoal Institute of Health, USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Jenopitk, Progress Gryphax, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck, USA</td>
			<td>107213</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#215; 10 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10035</td>
			<td></td>
		</tr>
		<tr>
			<td>90 &#215; 15 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Nikon, Japan</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickinson, USA</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans

Toksikologisk evaluering er afgørende for at forstå virkningerne af kemikalier på levende organismer i grundforskning og anvendt biologiske videnskab felter. En ikke-pattedyr jord runde orm, Caenorhabditis elegans, er en værdifuld model organisme for toksikologi undersøgelser på grund af dens bekvemmelighed og manglende Dyreetik problemer sammenlignet med pattedyr animalsk systemer. I denne protokol, er en detaljeret procedure for toksikologisk evaluering af kemikalier i C. elegans beskrevet. Et klinisk anticancer lægemiddel, etoposide, som er rettet mod menneskelige topoisomerase II og hæmmer DNA-replikation af menneskelige kræftceller, blev valgt som model test kemiske. Alder-synkroniseret C. elegans æg blev udsat for enten dimethylsulfoxid (DMSO) eller etoposide, og derefter vækst af C. elegans blev overvåget hver dag i 4 dage af stereo-mikroskop observation. Det samlede antal æg lagt fra C. elegans behandlet med DMSO, eller etoposide var også tælles ved hjælp af stereo-mikroskop. Etoposide behandling væsentligt berørt den vækst og reproduktion af C. elegans. Ved sammenligning af det samlede antal æg lagt fra orme med forskellige behandling perioder med kemikalier, kan det besluttes, at reproduktiv toksicitet af kemikalier på C. elegans reproduktion er reversibel eller irreversibel. Disse protokoller kan være nyttige for både udviklingen af forskellige stoffer og risikovurdering af miljømæssige giftstoffer.

Behandling af etoposide (24-96 h) betydeligt forsinket vækst af C. elegans. 96 h inkubation voksede etoposide-behandlet orme til 0,86 mm i kropslængde, mens ormene køretøj-behandlede voksede til 1,04 mm (figur 1). Væksthæmning var øjensynlig også observeret under stereo-mikroskop observation (figur 2). Vi begyndte at se æg fra ormene køretøj-behandlet på 72 h inkubation. På den anden side blev æg observeret...

Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

En grundlæggende protokol at evaluere toksiciteten af kemikalier i en model dyr, Caenorhabditis elegans, er beskrevet. Metoden er nem og nyttig for udviklingen af lægemidler samt for risikovurderingen af forskellige miljøgifte.

Måle effekten af kemikalier på vækst og reproduktion af Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

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Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

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