Name: a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro
Uploaded: 2017-05-17T13:00:00
Description: Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

Dette arbejde blev støttet af følgende tilskud: Naturvidenskabsfonden i Kina (nr. 81572198; nr. 81260161; nr. 81000460); Natural Science Foundation i Guangdong-provinsen, Kina (nr. 2015A030313772); Kina Postdoktorvidenskabelige Foundation Funded Project (nr. 2013M530385); Medical Research Foundation i Guangdong-provinsen, Kina (nr. A2016314); Shenzhen Videnskab og Teknologi Projekter (Nr. JCYJ20160301111338144; Nr. JSGG20151030140325149; Nr. JSGG20140519105550503; No.GJHZ20130412153906739; No. JCYJ20140414170821160; No.JCYJ20140414170821200).

Denne undersøgelse blev godkendt af Human Ethics Committee i Shenzhen Second People&#39;s Hospital. 1. Human articular cartilage Tissue Collection og chondrocyte Culture <ol><li> Fremstilling af materialer <ol><li> Forbered Dulbeccos modificerede ørnemedium (DMEM) medium suppleret med 10% føtalt bovint serum og 1% penicillin-streptomycin. Klargør 1 mg / ml collagenase II, 0,25% trypsin-EDTA, phosphatpufret saltopløsning (PBS) og en cellefilter (40 μm nylon). </li></o...

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W., 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=Exploring+Genome-wide+DNA+Methylation+Profiles+Altered+in+Kashin-Beck+Disease+Using+Infinium+Human+Methylation+450+Bead+Chips.">Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.</a> Biomed Environ Sci. 29 (7), 539-543 (2016).</li><li>Li, X. L., 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=Optimization+of+an+HPLC+Method+for+Determining+the+Genomic+Methylation+Levels+of+Taxus+Cells.">Optimization of an HPLC Method for Determining the Genomic Methylation Levels of Taxus Cells.</a> J Chromatogr Sci. 54 (2), 200-205 (2016).</li><li>Maghbooli, Z., 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=Global+DNA+methylation+as+a+possible+biomarker+fordiabetic+retinopathy.">Global DNA methylation as a possible biomarker fordiabetic retinopathy.</a> Diabetes Metab Res Rev. 31 (2), 183-189 (2015).</li><li>Barlic, A., Drobnic, M., Malicev, E., Kregar-Velikonja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+gene+expression+in+human+articular+chondrocytes+assigned+for+autologous+implantation.">Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation.</a> J Orthop Res. 26 (6), 847-853 (2008).</li><li>Legendre, F., 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=Enhanced+hyaline+cartilage+matrix+synthesis+in+collagen+sponge+scaffolds+by+using+siRNA+to+stabilizechondrocytes+phenotype+cultured+with+bone+morphogenetic+protein-2+under+hypoxia.">Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilizechondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.</a> Tissue Eng Part C Methods. 19 (7), 550-567 (2013).</li><li>Niethammer, T. 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Chondrocyt dedifferentiation in vitro alvorligt kompromitterer resultatet af ACI i behandlingen af ​​brusk fejl reparation 11 , 12 . For at optimere ACI-resultatet er det afgørende at undgå anvendelse af dedifferentierede chondrocytter 13 . Undersøgelser har antydet, at det generelle DNA-methyleringsniveau er forbundet med omfanget af chondrocyt dedifferentiation 4 , 6</...

Autolog chondrocytimplantation (ACI) er en forholdsvis ny, state-of-the-art procedure til behandling af ledbruskdefekter 1 , 2 . Et af de afgørende trin i ACI er amplifikation af chondrocytter via monolagskultur in vitro . Under amplifikation mister hyalinkondrocytterne let deres fænotype og bliver dedifferentierede, hvilket er uønsket for ACI-behandling 3 , 4 . For at optimere udfaldet af ACI-behandling bør omfanget af chondrocyt dedifferentiering bestemmes før genplantning. Det er afgørende at etablere en økonomisk og hurtig måde at bestemme kondrocytternes status på. For nylig har forbindelsen mellem DNA-methylering og chondrocyt dedifferentiering tiltrukket meget opmærksomhed 4 , 5 , 6 . DNA-methylering er en proces af whIch methylgrupper tilsættes til DNA, hvilket resulterer i omdannelsen af ​​cytosinrester til 5-methylcytosin (5-mC). For at belyse DNA-methylationens biologi ved chondrocyt dedifferentiering er det første trin at evaluere DNA-methyleringsniveauet af chondrocytter, som hidtil har vist sig at være udfordrende. Bisulfit genomisk sekventering er den mest anvendte teknik til analyse af DNA-methylering 7 , 8 . I denne analyse forårsager bisulfitomdannelse DNA-nedbrydning, og således skal der tilvejebringes en væsentlig mængde prøve til analysen. Højeffektive væskekromatografi (HPLC) har også været anvendt til at kvantificere globale DNA-methyleringsniveauer 9 , 10 . HPLC-analyse kræver imidlertid genomisk DNA-fordøjelse. Desuden kræves avancerede og dyre eksperimentelle instrumenter. Derfor ud over de høje omkostninger er disse eksperimentelle procedurer tidsforbruguming. Anti-5-mC antistoffer er nu blevet kommercielt tilgængelige, hvilket har skabt muligheden for immun blotting af 5-mC-holdigt genomisk DNA fra komplekse genomer. I denne rapport ekstraherede vi genomisk DNA fra chondrocytter dyrket i en række monolagskulturer. Vi brugte et dot blot assay til at evaluere 5-mC indholdet i humane chondrocytter med forskellige antal passager. Vi fandt ud af, at 5-mC indhold blev forøget i stærkt dedifferentierede chondrocytter sammenlignet med chondrocytter med lav grad dedifferentiering. Derudover identificerede vi et forhold mellem dedifferentiation status og 5-mC niveauer. Endelig rapporterede vi, at ændringerne i 5-mC-indhold var forbundet med chondrocytfænotypen. Derfor er 5-mC dot blot-analysen en pålidelig, enkel og hurtig metode til at detektere DNA-methyleringsniveauet i chondrocytter.

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			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>DMEM&#160;&#160;</td>
			<td>Gibco Inc.</td>
			<td>11965&#8211;092</td>
			<td>Warm in 37 &#176;C water bath before use</td>
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			<td rowspan="2">Phosphate-Buffered Saline(PBS)</td>
			<td rowspan="2">HyClone Inc.</td>
			<td rowspan="2">SH30256.01B</td>
			<td>D-PBS, free of&#160;</td>
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			<td>Ca2+/Mg2+</td>
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			<td>FBS</td>
			<td>Gibco Inc.</td>
			<td>10099-141</td>
			<td></td>
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		<tr>
			<td>0.25%Trypsin/EDTA</td>
			<td>Gibco Inc.</td>
			<td>25200-056</td>
			<td></td>
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			<td>1%Penicillin-Streptomycin</td>
			<td>Gibco Inc.</td>
			<td>15140-122</td>
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			<td>Chloroform</td>
			<td>Mallinckrodt</td>
			<td>4440</td>
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			<td>Isoamyl Alcohol</td>
			<td>Sigma</td>
			<td>I-3643</td>
			<td></td>
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			<td>Phenol</td>
			<td>Gibco BRL</td>
			<td>15513-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Gibco BRL</td>
			<td>24568-2</td>
			<td></td>
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			<td>TAE buffer&#160;</td>
			<td>Bio Whittaker</td>
			<td>16-011V</td>
			<td></td>
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			<td>Distilled Water</td>
			<td>Gibco BRL</td>
			<td>15230-170</td>
			<td></td>
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			<td>1 M Tris-HCl</td>
			<td>Biosharp Inc.</td>
			<td>BL514A</td>
			<td></td>
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			<td>Tween20</td>
			<td>Biotopped Inc.</td>
			<td>C58H114O26</td>
			<td></td>
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			<td>BSA</td>
			<td>Proliant Inc.</td>
			<td>68700</td>
			<td></td>
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			<td>Collagenase, Type II</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
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			<td>Names&#160;&#160;&#160;&#160;&#160;</td>
			<td>Company&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Catalog number&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td>Cell Strainer(40&#956;m nylon&#65289;</td>
			<td>FALCON Inc.</td>
			<td>352340</td>
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			<td>Hemocytometer</td>
			<td>ISOLAB Inc.</td>
			<td>075.03.001</td>
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			<td>Falcon 100 mm&#160; dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
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			<td>Centrifuge Tubes</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
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			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td></td>
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			<td>ThermoMixer</td>
			<td>MIULAB</td>
			<td>MTH-100</td>
			<td></td>
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			<td>Carbon dioxide cell incubator</td>
			<td>Thermo scientific</td>
			<td>3111</td>
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			<td>Chemi-imaging Analyse System</td>
			<td>UVITEC Cambridge</td>
			<td>ALLIANCE</td>
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a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro

Den dedifferentiering af hyalinkondrocytter i fibroblastiske chondrocytter følger ofte monolagsudvidelse af chondrocytter in vitro . Det globale DNA-methyleringsniveau for chondrocytter betragtes som en egnet biomarkør for tabet af chondrocytfænotypen. Resultater baseret på forskellige eksperimentelle metoder kan imidlertid være inkonsekvente. Derfor er det vigtigt at etablere en præcis, enkel og hurtig metode til at kvantificere globale DNA-methyleringsniveauer under chondrocyt dedifferentiering. Nuværende genomiske brede methyleringsanalyseteknikker afhænger i vid udstrækning af bisulfit genomisk sekventering. På grund af DNA-nedbrydning under bisulfit-omdannelse kræver disse fremgangsmåder typisk et stort prøvevolumen. Andre metoder anvendt til at kvantificere globale DNA-methyleringsniveauer indbefatter højtydende væskekromatografi (HPLC). HPLC kræver imidlertid fuldstændig fordøjelse af genomisk DNA. Derudover er de uforholdsmæssigt høje omkostninger ved HPLC iStruments begrænser HPLCs bredere anvendelse. I dette studie blev genomisk DNA (gDNA) ekstraheret fra humane chondrocytter dyrket med varierende antal passager. GDNA-methyleringsniveauet blev detekteret under anvendelse af et methyleringsspecifik dot-blot-assay. I denne dot blot-tilgang blev en gDNA-blanding indeholdende det methylerede DNA, der skal detekteres, plettet direkte på en N + -membran som en prikk inde i et tidligere udtrukket cirkulært template-mønster. Sammenlignet med andre gelelektroforese-baserede blottingsmetoder og andre komplekse blottingsprocedurer sparer dot blot-metoden signifikant tid. Dot blots kan desuden detektere det overordnede DNA-methyleringsniveau ved anvendelse af et kommercielt tilgængeligt 5-mC antistof. Vi fandt ud af, at DNA-methyleringsniveauet varierede mellem monolag-subkulturerne og kunne derfor spille en central rolle i chondrocyt dedifferentiering. 5-mC dot blot er en pålidelig, enkel og hurtig metode til at detektere det generelle DNA-methyleringsniveau for at evaluereE chondrocytfænotype.

Chondrocytter blev dyrket i et monolag op til passage 6 (P6). Chondrocytter udviste progressive fænotypiske ændringer med successive passager i monolagskulturen. P0-chondrocytemorfologien var runde, mens cellerne blev stærkt klæbet og fladt med successive passager op til P6 ( Figur 1 ). Denne morfologiske forandring er typisk for chondrocyt dedifferentieringsprocessen. I mellemtiden viste resultaterne af varmekortet, at det generelle methyleringsniveau for CpG-steder...

Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Vi præsenterer en metode til kvantificering af DNA-methylering baseret på 5-methylcytosin (5-mC) dot blot. Vi fastslog 5-mC niveauerne under chondrocyt dedifferentiering. Denne enkle teknik kunne bruges til hurtigt at bestemme chondrocytfænotypen ved ACI-behandling.

En 5-mC prikkeblot-analyse, der kvantificerer DNA-methyleringsniveauet for chondrocyt-differentiering<em&gt; In Vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

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A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

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