Glial Fibrillary Acidic Protein as Biomarker Indicates Purity and Property of Auricular Chondrocytes
Glial Fibrillary Acidic Protein as Biomarker Indicates Purity and Property of Auricular Chondrocytes
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DOI：
10.1089/biores.2019.0058
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发表时间：
2020-03
影响因子：
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通讯作者：
Satoru Nishizawa;Sanshiro Kanazawa;Y. Fujihara;Y. Asawa;S. Nagata;Motohiro Harai;Atsuhiko Hikita;T. Takato;K. Hoshi
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文献类型：
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作者：
Satoru Nishizawa;Sanshiro Kanazawa;Y. Fujihara;Y. Asawa;S. Nagata;Motohiro Harai;Atsuhiko Hikita;T. Takato;K. Hoshi
取代以前用于修复和重建因事故和疾病而失去的耳廓和鼻子的硅胶植入物，一种使用组织工程软骨的新治疗方法引起了人们的关注。在这种方法中，培养细胞的质量很重要，因为它影响治疗结果。然而，软骨细胞，特别是耳软骨细胞的标记物尚未建立。本研究的目的是建立一个评价耳廓软骨细胞作为再生软骨组织细胞来源的最佳标记物。采用基因芯片技术对未分化和去分化耳廓软骨细胞的基因表达水平进行综合比较，寻找在分化细胞中高表达而在去分化细胞中表达显著降低的候选质控指标。我们发现，在耳廓软骨细胞中，胶质纤维酸性蛋白(GFAP)是一种随着连续传代而减少的标志物。在关节软骨细胞、肋软骨细胞和成纤维细胞中未检测到GFAP，这些细胞需要在细胞培养中与耳软骨细胞区分开来。在培养的耳软骨细胞中观察到GFAP mRNA的表达，在细胞裂解液和培养上清液中也检测到GFAP蛋白的水平。然而，从细胞裂解产物中检测到的GFAP水平随着孵育期的增加而显著降低。相反，细胞培养上清液中的蛋白质含量不受孵育时间的影响。此外，培养细胞上清液中GFAP的蛋白水平与这些细胞在体外和体内产生软骨基质有关。通过测定耳廓软骨细胞培养上清液中GFAP蛋白水平，可以预测耳廓软骨细胞软骨基质的生成能力。因此，GFAP被认为是培养的耳软骨细胞纯度和特性的标志。
Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.