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

这项工作得到以下赠款的支持：中国自然科学基金（No. 81572198; 81260161; No. 81000460）;中国广东省自然科学基金（编号：2015A030313772）;中国博士后科学基金资助项目（No. 2013M530385）;中国广东省医学研究基金（No. A2016314）;深圳市科技项目（No.JCYJ20160301111338144; No.JSGG20151030140325149; No.JSGG20140519105550503; No.GJHZ20130412153906739; No.JCYJ20140414170821160; No.JCYJ20140414170821200）。

本研究由深圳市第二人民医院人类伦理委员会批准。 人关节软骨组织收集和软骨细胞培养<ol><li> 材料的制备 <ol><li>制备补充有10％胎牛血清和1％青霉素 - 链霉素的Dulbecco改良的Eagle培养基（DMEM）培养基。制备1mg / mL胶原酶II，0.25％胰蛋白酶-EDTA，磷酸盐缓冲盐水（PBS）和细胞过滤器（40μm尼龙）。 </li></ol></li><li> 人关节软骨组织收集 <ol><li>创伤后供体...

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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. R., 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=Analysis+of+the+autologous+chondrocyte+quality+of+matrix-based+autologous+chondrocyte+implantation+in+the+knee+joint.">Analysis of the autologous chondrocyte quality of matrix-based autologous chondrocyte implantation in the knee joint.</a> Int Orthop. 40 (1), 205-212 (2016).</li><li>Hayatsu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bisulfite+genomic+sequencing+used+in+the+analysis+of+epigenetic+states,+a+technique+in+the+emerging+environmental+genotoxicology+research.">The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research.</a> Mutat Res. 659 (1-2), 77-82 (2008).</li><li>Haque, N., Nishiguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+sequencing+for+cytosine-methylation+analysis+in+plants.">Bisulfite sequencing for cytosine-methylation analysis in plants.</a> Methods Mol Biol. 744, 187-197 (2011).</li><li>Ananiev, G. 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软骨细胞去分化在体外严重损害ACI治疗软骨缺损修复的结果11,12 。为了优化ACI结果，避免使用去分化软骨细胞13至关重要。研究表明，一般的DNA甲基化水平与软骨细胞去分化的程度有关。因此，在临床应用前，建立可靠，快速的方法来检测软骨细胞的一般DNA甲基化状态。 为了检测去分化的人软骨细胞中的一般DNA甲基化水平，我...

自体软骨细胞植入（ACI）是一种相对较新的，最先进的手术来治疗关节软骨缺损1,2 。 ACI的关键步骤之一是通过单层培养体外扩增软骨细胞 。在扩增期间，透明软骨细胞容易失去其表型并变得去分化，这对于ACI治疗3,4是不合需要的。为了优化ACI治疗的结果，软骨细胞去分化的程度应在再植之前确定。建立经济，快速的方法来确定软骨细胞的状态是至关重要的。最近，DNA甲基化与软骨细胞去分化之间的关联已经引起了很多关注4，5，6 。 DNA甲基化是一个过程将甲基添加到DNA中，导致胞嘧啶残基转化为5-甲基胞嘧啶（5-mC）。 为了阐明软骨细胞去分化中DNA甲基化的生物学，第一步是评估软骨细胞的DNA甲基化水平，迄今证明是有挑战性的。亚硫酸氢盐基因组测序是分析DNA甲基化最广泛使用的技术7,8 。在该测定中，亚硫酸氢盐转化引起DNA降解，因此必须提供大量的样品用于测定。此外，高效液相色谱（HPLC）已被用于量化全球DNA甲基化水平9,10 。然而，HPLC分析需要基因组DNA消化。此外，需要先进和昂贵的实验仪器。因此，除了高成本之外，这些实验程序是时间短的uming。抗-5-mC抗体现在已经成为可商购的，这已经产生了来自复杂基因组的含有5-mC的基因组DNA的免疫印迹的可能性。 在本报告中，我们从一系列单层培养物中生长的软骨细胞中提取基因组DNA。我们使用斑点印迹测定来评估具有不同数量通道的人软骨细胞中的5-mC含量。我们发现，与低级去分化的软骨细胞相比，高度去分化的软骨细胞中5-mC含量增加。此外，我们确定了去分化状态和5-mC水平之间的关系。最后，我们报道了5-mC含量的变化与软骨细胞表型有关。因此，5-mC斑点印迹测定是检测软骨细胞中DNA甲基化水平的可靠，简单和快速的方法。

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			<td>Reagents</td>
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			<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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		<tr>
			<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>
		</tr>
		<tr>
			<td>1%Penicillin-Streptomycin</td>
			<td>Gibco Inc.</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Mallinckrodt</td>
			<td>4440</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamyl Alcohol</td>
			<td>Sigma</td>
			<td>I-3643</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>TAE buffer&#160;</td>
			<td>Bio Whittaker</td>
			<td>16-011V</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Gibco BRL</td>
			<td>15230-170</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Proliant Inc.</td>
			<td>68700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td>Comments</td>
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			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Cell Strainer(40&#956;m nylon&#65289;</td>
			<td>FALCON Inc.</td>
			<td>352340</td>
			<td></td>
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		<tr>
			<td>Hemocytometer</td>
			<td>ISOLAB Inc.</td>
			<td>075.03.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 100 mm&#160; dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
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		<tr>
			<td>Centrifuge Tubes</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>MIULAB</td>
			<td>MTH-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide cell incubator</td>
			<td>Thermo scientific</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi-imaging Analyse System</td>
			<td>UVITEC Cambridge</td>
			<td>ALLIANCE</td>
			<td></td>
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a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro

透明软骨细胞去分化成纤维细胞软骨细胞通常伴随体外软骨细胞的单层扩张。软骨细胞的全球DNA甲基化水平被认为是软骨细胞表型丧失的合适的生物标志物。然而，基于不同实验方法的结果可能不一致。因此，建立一种精确，简单，快速的方法来量化软骨细胞去分化中的全局DNA甲基化水平是非常重要的。 目前的全基因组甲基化分析技术主要依靠亚硫酸氢盐基因组测序。由于亚硫酸氢盐转化期间的DNA降解，这些方法通常需要较大的样品体积。用于定量全球DNA甲基化水平的其他方法包括高效液相色谱（HPLC）。然而，HPLC需要完全消化基因组DNA。另外，HPLC的成本高昂结构限制了HPLC的广泛应用。 在本研究中，从用不同数量传代培养的人软骨细胞中提取基因组DNA（gDNA）。使用甲基化特异性斑点印迹法检测gDNA甲基化水平。在该斑点印迹方法中，将含有待检测的甲基化DNA的gDNA混合物直接点在N +膜上，作为先前绘制的圆形模板图案内的点。与其他基于凝胶电泳的印迹方法和其他复杂印迹方法相比，斑点印迹法节省了大量时间。此外，斑点印迹可以使用市售的5-mC抗体检测总DNA甲基化水平。我们发现DNA甲基化水平在单层亚文化之间不同，因此可能在软骨细胞去分化中起关键作用。 5-mC斑点印迹是检测一般DNA甲基化水平进行评估的可靠，简单和快速的方法e软骨细胞表型。

软骨细胞在单层培养直至第6代（P6）。软骨细胞随着单层培养物的连续传代显示进行性表型变化。 P0软骨细胞形态是圆形的，而细胞变成高度夹持和平坦化，连续传代直到P6（ 图1 ）。这种形态变化是软骨细胞去分化过程的典型。同时，热图的结果表明CpG位点的一般甲基化水平随着传代培养的增加而增加。 5-mC斑点印迹分析进一步表明，较高的CpG甲基化水平伴?...

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

我们提出了一种基于5-甲基胞嘧啶（5-mC）斑点印迹定量DNA甲基化的方法。我们在软骨细胞去分化中测定了5-mC的水平。这种简单的技术可用于快速测定ACI治疗中的软骨细胞表型。

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</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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Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...