Jia Zhaofeng, Liang Yujie, Ma Bin, Xu Xiao, Xiong Jianyi, Duan Li, Wang Daping
Guangzhou Medical University; Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University).
Department of Chemistry, The Chinese University of Hong Kong.
J Vis Exp. 2017 May 17(123):55565. doi: 10.3791/55565.
The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.
在体外培养过程中，透明软骨细胞向成纤维细胞样软骨细胞的去分化常伴随着软骨细胞的单层扩增。软骨细胞的整体DNA甲基化水平被认为是软骨细胞表型丧失的合适生物标志物。然而，基于不同实验方法得到的结果可能不一致。因此，建立一种精确、简单且快速的方法来量化软骨细胞去分化过程中的整体DNA甲基化水平具有重要意义。目前全基因组甲基化分析技术很大程度上依赖于亚硫酸氢盐基因组测序。由于亚硫酸氢盐转化过程中DNA会发生降解，这些方法通常需要大量样本。其他用于量化整体DNA甲基化水平的方法包括高效液相色谱法（HPLC）。然而，HPLC需要对基因组DNA进行完全消化。此外，HPLC仪器高昂的成本限制了其更广泛的应用。在本研究中，从不同传代次数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。在这种斑点印迹方法中，将含有待检测甲基化DNA的gDNA混合物直接点样到N膜上，形成预先绘制的圆形模板图案内的一个点。与其他基于凝胶电泳的印迹方法和其他复杂的印迹程序相比，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抗体检测总体DNA甲基化水平。我们发现单层传代培养物之间的DNA甲基化水平存在差异，因此可能在软骨细胞去分化中起关键作用。5 - mC斑点印迹法是一种可靠、简单且快速的检测一般DNA甲基化水平以评估软骨细胞表型的方法。
