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本实验为建立永生化的猪耳软骨细胞系并探讨它的生物学特性,以包含人端粒酶逆转录酶催化亚基(human telomerase reverse transcriptase,hTERT)基因的逆转录病毒载体(pBABE-hTERT)转染至正常猪耳软骨细胞中。经筛选、挑取克隆,得永生化软骨细胞系(TL1),在体外已连续培养10个月。RT-PCR和PCR-ELISA法测得其有高水平的端粒酶稳定表达。通过生长曲线、HE染色、流式细胞仪分析,以及裸鼠成瘤实验,表明它的细胞增殖能力增加,凋亡率下降并维持在体外培养早期细胞的水平,且细胞无恶变迹象。对软骨特异性分子的RT-PCR、Western杂交和免疫组化的结果分析,发现TL1中有SOX9的表达;无结构完整的aggrecan表达;有低水平的Ⅱ型胶原表达;Ⅰ型胶原的表达随细胞的表型改变而改变。综上所述,通过外源性hTERT的稳定表达,建立了永生化的猪耳软骨细胞系;但是它在单层培养的状态下,软骨细胞特异性的基因表达逐步发生改变,导致其部分丧失软骨细胞的正常表型。++
In order to establish an immortalized porcine ear cartilage cell line and to explore its biological characteristics, a retroviral vector containing human telomerase reverse transcriptase (hTERT) gene (pBABE-hTERT ) Were transfected into normal porcine ear cartilage cells. After screening, the clones were picked to obtain immortalized chondrocytes (TL1) and cultured continuously in vitro for 10 months. The results of RT-PCR and PCR-ELISA showed that there was high level of telomerase stable expression. The growth curve, HE staining, flow cytometry analysis and tumorigenesis in nude mice showed that the cell proliferation ability increased, the apoptosis rate decreased and maintained at the level of early culture cells in vitro, and the cells showed no signs of malignancy. Analysis of the results of RT-PCR, Western blot and immunohistochemistry of cartilage-specific molecules showed that there was SOX9 expression in TL1; no aggrecan expression was found; low expression of type II collagen was observed; the expression of type I collagen The phenotype of the cell changes. In summary, the immortalized porcine ear cartilage cell line was established by stable expression of exogenous hTERT; however, in monolayer culture, chondrocyte-specific gene expression was gradually changed, resulting in a partial loss of hTERT The normal phenotype of chondrocytes. ++