Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer
Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer
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DOI：
10.1007/s00441-005-0101-4
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发表时间：
2006-04-01
影响因子：
3.6
通讯作者：
Akamine, A
中科院分区：
文献类型：
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作者：
Fujii, S;Maeda, H;Akamine, A
牙周膜是连接牙骨质和牙槽骨的高度特化组织，影响牙齿的寿命。然而，由于缺乏特异性的标记物和细胞系，人们对PDL细胞的确切特性和再生机制知之甚少。因此，本研究旨在通过猴病毒40 T抗原（SV 40 T-Ag）和人端粒酶逆转录酶（hTERT）转染，建立3株永生化人PDL成纤维细胞系，以期获得具有原代细胞特性的细胞。将转染的细胞命名为STPLF。通过半定量逆转录聚合酶链反应（RT-PCR）方法、拉伸PCR分析或Western印迹分析验证所有STPLF系中SV 40 T-Ag和hTERT的表达。所有的STPLF在超过120个群体倍增（PD）时显示稳定的增殖，而原代人PDL成纤维细胞（HPLF）在10-20个PD时停止。通过RT-PCR分析的表征显示，所有STPLF基因模拟其各自的原始HPLF基因的表达。STPLF表达了侏儒相关转录因子-2、osterix、碱性磷酸酶、骨桥蛋白、骨钙蛋白、骨膜蛋白、NF-κ B配体受体激活因子、骨保护素、表皮生长因子受体、α-平滑肌肌动蛋白和XII型胶原。用50 μ g/ml抗坏血酸和2 mM β-甘油磷酸盐刺激STPLF 4周比用相同试剂培养的HPLF产生更多的钙化沉积物。这些结果表明，每个STPLF线保留了各自的原始HPLF的特性，STPLF获得了增加的钙化活性，并且STPLF是用于研究人类PDL的生物学和再生机制的有用工具。
The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 mu g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.