背景:在软骨再生医学中,移植的软骨细胞含有种群的混合物,使均匀软骨组织的再生复杂化。我们小组先前报道,通过选择快速生长的细胞可以富集具有较高软骨形成能力的软骨细胞。在这项研究中,研究了快速生长的软骨细胞的详细特性,并将其与缓慢生长的细胞进行了比较。
方法:用羧基荧光素琥珀酰亚胺酯(CFSE)对人耳软骨细胞进行荧光标记,并使用流式细胞仪进行分析,根据前向散射和侧向散射,聚焦于荧光强度和细胞形态所指示的分裂速率。在CFSE标记后的第2天和第4天收获快速和缓慢生长的细胞组,并检查了它们在体外产生软骨基质的能力。为了比较体内的软骨形成能力,将细胞接种在聚乳酸支架上并移植到裸鼠体内。通过微阵列分析研究了快速细胞组和慢细胞组之间的基因表达差异。
结果:在CFSE标签后的第2天,生长迅速的细胞群增殖率最高。颗粒培养结果表明,快速细胞组比慢细胞组每个细胞产生更多的糖胺聚糖。在CFSE标记后第2天,快速细胞组中糖胺聚糖的产生量最高,表明软骨形成能力高。此外,微阵列,基因本体论,和京都百科全书的基因和基因组途径分析显示,促进细胞分裂的基因如起源识别复合物亚基1上调和抑制细胞分裂的基因如细胞周期蛋白依赖性激酶抑制剂1A下调。除了细胞周期相关基因,软骨细胞相关基因,如serpin家族B成员2,簇蛋白,骨形态发生蛋白2和基质金属蛋白酶3下调,而参与干细胞维持的成纤维细胞生长因子5,以及含有2A的卷曲螺旋和C2结构域,这是纤毛形成所必需的,被上调了。
结论:结果表明,快速细胞组增殖良好,具有更多的未分化特性,暗示更高的干性。本发现为快速细胞群在软骨再生中的应用提供了基础。
BACKGROUND: In cartilage regenerative medicine, transplanted chondrocytes contain a mixture of populations, that complicates the regeneration of uniform cartilage tissue. Our group previously reported that chondrocytes with higher chondrogenic ability could be enriched by selection of rapidly growing cells. In this study, the detailed properties of rapidly growing chondrocytes were examined and compared to slowly growing cells.
METHODS: Human auricular chondrocytes were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and analyzed using flow cytometry, focusing on division rates as indicated by fluorescence intensity and cell morphology according to the forward scatter and side scatter. Rapid and slow growing cell groups were harvested on days 2 and 4 after CFSE labeling, and their ability to produce cartilage matrix in vitro was examined. To compare the chondrogenic ability in vivo, the cells were seeded on poly-l-lactic acid scaffolds and transplanted into nude mice. Gene expression differences between the rapid and slow cell groups were investigated by microarray analysis.
RESULTS: On day 2 after CFSE labeling, the rapidly growing cell group showed the highest proliferation rate. The results of pellet culture showed that the rapid cell group produced more glycosaminoglycans per cell than the slow cell group. The amount of glycosaminoglycan production was highest in the rapid cell group on day 2 after CFSE labeling, indicating high chondrogenic ability. Furthermore, microarray, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed upregulation of genes that promote cell division such as origin recognition complex subunit 1 and downregulation of genes that inhibit cell division such as cyclin dependent kinase inhibitor 1A. Besides cell cycle-related genes, chondrocyte-related genes such as serpin family B member 2, clusterin, bone morphogenetic protein 2, and matrix metalloproteinase 3 were downregulated, while fibroblast growth factor 5 which is involved in stem cell maintenance, and coiled-coil and C2 domain containing 2A, which is required for cilia formation, were upregulated.
CONCLUSIONS: The results showed that the rapid cell group proliferated well and had more undifferentiated properties, suggesting a higher stemness. The present findings provide a basis for the use of the rapid cell group in cartilage regeneration.