Bodine P V, Trailsmith M, Komm B S
Women's Health Research Institute, Wyeth-Ayerst, Radnor, Pennsylvania, USA.
J Bone Miner Res. 1996 Jun;11(6):806-19. doi: 10.1002/jbmr.5650110612.
Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.
目前有多种成骨细胞系在使用，但这些细胞系在与成年人类生物学和疾病的相关性方面，或者在用于生化和分子分析的适用性方面存在局限性。因此，我们着手开发条件性转化的成年人类成骨细胞系。成骨细胞取自正常的外植松质骨芯片培养物。这些细胞用编码温度敏感型大T抗原突变体的腺病毒-ori-SV40 tsA 209进行感染。用这种病毒永生化的细胞在34℃的允许温度下表现出转化表型，但在40℃的非允许温度下恢复为正常表型。使用这种方法，我们分离出了几个细胞克隆，并描述了其中一个命名为HOB-02-C1的克隆的特性。免疫细胞化学显示，超过95%的细胞在两个温度下都表达大T抗原。这些细胞在34℃时呈指数增殖，倍增时间约为2天，但在40℃时不可逆地停止分裂。然而，当细胞在较高温度下维持6天时，细胞体积增加超过2倍。该克隆在两个温度下均表达α1（I）型前胶原mRNA并分泌I型前胶原C肽，尽管在40℃时水平略有升高。该细胞系在34℃时表达碱性磷酸酶活性，该酶的基础水平在40℃时增加2至6倍。在两个温度下，1α,25-二羟基维生素D3（维生素D3）均可诱导碱性磷酸酶活性增加4至8倍，但转化生长因子-β1（TGF-β1）在40℃时可抑制该酶表达超过90%。当克隆在34℃维持时，维生素D3还可诱导骨钙素分泌增加10倍，在40℃时这种诱导作用增强超过8倍。甲状旁腺激素和福司可林在34℃时可刺激细胞内细胞环磷酸腺苷（cAMP）产生增加4至6倍，在40℃时这种刺激作用增强2至4倍。相比之下，前列腺素E2仅在细胞维持在34℃时可刺激cAMP增加7至8倍。该细胞系在34℃时分泌TGF-β1和白细胞介素-6（IL-6），但在40℃时只有IL-6的基础分泌增加70%。最后，茜素红-S组织化学染色表明，这些细胞在两个温度下均产生矿化结节。总之，本研究结果表明HOB-02-C1细胞具有成熟的成骨细胞表型。因此，这个新的细胞系以及以类似方式获得的其他细胞系，应该是用于成年人类成骨细胞生物学细胞、生化和分子研究的有价值的体外工具。
