Abstract—

A new nonimmortalized cell line (MSC-GING) was isolated from the gingiva of a 35-year-old healthy donor and characterized. The analysis of different properties was carried out at the 6th, 7th, 13th, 18th, 20th, and 23rd passages. During long-term cultivation, the proportion of aging cells according to the activity of β-galactosidase increased. The cloning efficiency of MSC-GING cells was significantly reduced, and the number of aging β-galactosidase-positive cells was gradually increased during cell prolonged cultivation. Growth curves showed active cell proliferation at the sixth passage. It was significantly declined by the 18th and 20th passages. Karyotypic analysis at the 7th and 18th passages showed the presence of a diploid number of chromosomes (46) in metaphase plates. At passage 7, however, the proportion of cells with a normal diploid karyotype 46, XX, was 50 ± 5%. Other cells had a clonal rearrangement inv (10) (q21.1q25.1), an inversion of the long arm of chromosome 10. Their number significantly decreased during cultivation up to passage 18, while the proportion of cells with a normal diploid karyotype increased. The cell number with surface antigens common for human mesenchymal stem cells (CD44, CD73, CD90, CD105, HLA-ABC) and vimentin was high at passages 6 and 20, while CD34, CD45, and HLA-DR positive cells were very rare. It is noteworthy that no cells carrying markers of undifferentiated embryonic stem cells (Oct-4, SSEA-4, and SOX2) are observed at passages 6 and 20. It was shown that MSC-GING cells were able to differentiate in osteogenic and chondrogenic directions. The ability to differentiate in the adipogenic direction was manifested only at the level of glut4 gene expression. Induction of neuronal differentiation increased the expression the nse gene (neurospecific elonase). In general, the results confirmed the MSC status for the obtained line, but revealed significant karyotypic instability in the early passages, which decreased during the replicative aging.

中文翻译：

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人牙龈间充质干细胞的分离与鉴定

摘要-

从35岁健康供体的牙龈中分离出一种新的永生细胞系（MSC-GING）并进行了表征。在第6、7、13、18、20和23代进行了不同特性的分析。在长期培养期间，根据β-半乳糖苷酶的活性，衰老细胞的比例增加。在长时间培养过程中，MSC-GING细胞的克隆效率明显降低，衰老的β-半乳糖苷酶阳性细胞数量逐渐增加。生长曲线显示在第六代活跃细胞增殖。到第18和20代时，它显着下降。在第7和18代的核型分析表明，在中期板中存在二倍体数的染色体（46）。但是，在第7段，具有正常二倍体核型46 XX的细胞比例为50±5％。其他细胞具有克隆重排inv（10）（q21.1q25.1），是10号染色体长臂的倒置。在培养至18代之前，它们的数量显着减少，而具有正常二倍体核型的细胞比例增加。在第6和20代时，人间充质干细胞（CD44，CD73，CD90，CD105，HLA-ABC）和波形蛋白常见的具有表面抗原的细胞数量很高，而CD34，CD45和HLA-DR阳性细胞非常少见。值得注意的是，在第6和20代中未观察到带有未分化胚胎干细胞（Oct-4，SSEA-4和SOX2）标记的细胞。表明MSC-GING细胞能够在成骨和软骨形成方向分化。glut4基因表达。神经元分化的诱导表达增加的NSE基因（neurospecific elonase）。一般而言，结果证实了所获得品系的MSC状态，但揭示了早期传代中明显的核型不稳定性，在复制性衰老过程中有所降低。