Hamada Takashi, Sakai Tadahiro, Hiraiwa Hideki, Nakashima Motoshige, Ono Yohei, Mitsuyama Hirohito, Ishiguro Naoki
Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Nagoya J Med Sci. 2013 Feb;75(1-2):101-11.
Autologous chondrocyte implantation (ACI) is a method of cartilage repair. To improve the quality of regenerated tissue by ACI, it is essential to identify surface marker expression correlated with the differentiation status of monolayer expanded human articular chondrocytes and to define the index for discriminating dedifferentiated cells from monolayer expanded human articular chondrocytes. Normal human articular chondrocytes were cultured in monolayer until passage 4. At each passage, mRNA expression of collagen type I, II, and X and aggrecan was analyzed by real-time quantitative PCR, and the surface marker expression of CD14, CD26, CD44, CD49a, CD49c, CD54, and CD151 was analyzed by fluorescence-activated cell sorting (FACS). The ratios of mRNA levels of collagen type II to I (Col II/Col I) represented the differentiation status of chondrocytes more appropriately during monolayer culture. The surface marker expression of CD44, CD49c, and CD151 was upregulated according to the dedifferentiation status, whereas that of CD14, CD49a, and CD54 was downregulated. The most appropriate combination of the ratio of Col II/Col I was CD54 and CD44. Cell sorting was performed using a magnetic cell sorting system (MACS) according to CD54 and CD44, and real-time quantitative PCR was performed for the cell subpopulations before and after cell sorting. The expression of collagen type II and aggrecan of the chondrocytes after MACS was higher than that before sorting, but not significantly. The mean fluorescence intensity (MFI) ratio of CD54 to CD44 could be an adequate candidate as the index of the differentiation status.
自体软骨细胞移植（ACI）是一种软骨修复方法。为了提高ACI再生组织的质量，识别与单层扩增的人关节软骨细胞分化状态相关的表面标志物表达，并确定区分单层扩增的人关节软骨细胞中去分化细胞的指标至关重要。将正常人关节软骨细胞进行单层培养直至第4代。在每一代，通过实时定量PCR分析Ⅰ型、Ⅱ型和X型胶原蛋白以及聚集蛋白聚糖的mRNA表达，并通过荧光激活细胞分选（FACS）分析CD14、CD26、CD44、CD49a、CD49c、CD54和CD151的表面标志物表达。在单层培养过程中，Ⅱ型胶原蛋白与Ⅰ型胶原蛋白的mRNA水平之比（Col II/Col I）更能恰当地代表软骨细胞的分化状态。CD44、CD49c和CD151的表面标志物表达根据去分化状态上调，而CD14、CD49a和CD54的表达下调。Col II/Col I比值与CD54和CD44的组合最为合适。根据CD54和CD44使用磁性细胞分选系统（MACS）进行细胞分选，并对细胞分选前后的细胞亚群进行实时定量PCR。MACS分选后软骨细胞中Ⅱ型胶原蛋白和聚集蛋白聚糖的表达高于分选前，但差异不显著。CD54与CD44的平均荧光强度（MFI）比值可以作为分化状态指标的合适候选者。
