Xiaoxue Yin, Zhongqiang Chen, Zhaoqing Guo, Gengting Dang, Qingjun Ma, Shenwu Wang
Department of Orthopaedics, Peking University Third Hospital, Haidian District, Beijing 100083, China.
Biochem Biophys Res Commun. 2004 Mar 12;315(3):643-51. doi: 10.1016/j.bbrc.2004.01.102.
In the current study, in order to establish an immortalized osteoblast cell line, human mesenchymal stem cells (hMSCs) had been inducted into osteoblasts directionally by an osteo-inductive conditioned medium, then the osteoblasts were steadily transduced by a retroviral vector containing human telomerase reverse transcriptase (hTERT) gene. The expression of hTERT, the telomerase activity, the telomere lengths, the tumorigenesis and the osteogenesis characteristics of transduced cells at different population doublings (PDs) and the primary normal human osteoblast (hOB) were identified. The results demonstrated that hTERT gene had been transferred into human osteoblasts successfully; the transduced cell line-clone5 expressed telomerase activity and divided vigorously and now have undergone more than 120 PDs; The telomere length of clone5 elongated and was stable; Different eras of clone5 (PD 40 and PD 88) both expressed bone-specific markers, such as alkaline phosphatase (ALP), collagen type I, and osteopontin. And the quantitative assay of ALP activity showed that there were no significant differences among untransduced cells, PD 40 and PD 88 clone5 cells. Furthermore, the immortalized cell line was benign in nude mice tumor formation and soft agar colony formation assay.
在本研究中，为建立永生化成骨细胞系，人骨髓间充质干细胞（hMSCs）经成骨诱导条件培养基定向诱导为成骨细胞，然后用含人端粒酶逆转录酶（hTERT）基因的逆转录病毒载体稳定转导成骨细胞。鉴定了转导细胞在不同群体倍增数（PDs）时的hTERT表达、端粒酶活性、端粒长度、致瘤性和成骨特性，以及原代正常人成骨细胞（hOB）。结果表明，hTERT基因已成功转入人成骨细胞；转导的细胞系clone5表达端粒酶活性，增殖旺盛，现已经历超过120次PDs；clone5的端粒长度延长且稳定；clone5的不同代（PD 40和PD 88）均表达骨特异性标志物，如碱性磷酸酶（ALP）、I型胶原和骨桥蛋白。ALP活性的定量测定表明，未转导细胞、PD 40和PD 88 clone5细胞之间无显著差异。此外，该永生化细胞系在裸鼠肿瘤形成和软琼脂集落形成试验中表现为良性。
