永生化软骨细胞的生物学特性

BIOLOGICAL CHARACTERIZATION OF IMMORTALIZED PORCINE CHONDROCYTES BY INTRODUCTION OF TELOMERASE

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。

The aim of this project was to acquire a clonal immortalized porcine chondrocyte cell line and to determine its biological characterization. Porcine auricle chondrocytes were infected with a retroviral vector pBABE-hTERT followed by selection with 2μg/ml puromycin. RT-PCR and PCR-ELISA performed on separated colonies showed a cell line(TL1) with high and stable ectopic expression of hTERT. The growth curve, HE staining, flow cytometry, and tumorigenicity were analyzed to define the biological characterization of the immortalized chondrocytes. TL1 cells showed an increase in cellular viability and the reduction in apoptosis. But there was no malignant transformation and tumor development in nude mice. The changes of chondrocyte marker expression in this cell line revealed that it remained unstable phenotype. So high ectopic expression of hTERT is able to extend the life span of chondrocytes, but it unable to keep their differentiated phenotype during serial monolayer culture in vitro.