Li Qing, Liu Wei, Chen Jin Jun, Lu Jun Hong, Liu De Li, Cao Yi Lin
Department of Plastic Surgery, Ninth People's Hospital, Shanghai Second Medical University, Shanghai Key Laboratory of Tissue Engineering, Shanghai 200011.
Shi Yan Sheng Wu Xue Bao. 2003 Dec;36(6):435-44.
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 2 micrograms/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.
本项目的目的是获得一株克隆永生化猪软骨细胞系并确定其生物学特性。用逆转录病毒载体pBABE-hTERT感染猪耳廓软骨细胞，随后用2微克/毫升嘌呤霉素进行筛选。对分离的菌落进行的RT-PCR和PCR-ELISA显示，有一个细胞系（TL1）具有hTERT的高且稳定的异位表达。分析生长曲线、苏木精-伊红染色、流式细胞术和致瘤性以确定永生化软骨细胞的生物学特性。TL1细胞显示细胞活力增加且凋亡减少。但裸鼠中未发生恶性转化和肿瘤形成。该细胞系中软骨细胞标志物表达的变化表明其表型仍不稳定。因此，hTERT的高异位表达能够延长软骨细胞的寿命，但在体外连续单层培养过程中无法保持其分化表型。
