Development and characterization of a conditionally transformed adult human osteoblastic cell line
Development and characterization of a conditionally transformed adult human osteoblastic cell line
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条件转化成人成骨细胞系的开发和表征
DOI：
10.1002/jbmr.5650110612
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发表时间：
1996
影响因子：
6.2
通讯作者：
B. Komm
中科院分区：
文献类型：
--
作者：
P. Bodine;M. Trailsmith;B. Komm
许多成骨细胞系目前正在使用中，但这些细胞系在其与成人生物学和疾病的相关性方面或在其适用于生化和分子分析方面具有局限性。因此，我们进行了条件转化成人成骨细胞系的发展。成骨细胞从正常的植块松质骨片培养物中获得。这些细胞被腺病毒-ori-SV 40 tsA 209感染，该病毒编码温度敏感性大T抗原突变体。用该病毒永生化的细胞在34°C的允许温度下表达转化的表型，但在40°C的非允许温度下恢复正常表型。使用这种方法，我们分离了几个细胞克隆，并描述了一个被命名为HOB-02-C1的细胞克隆的特征。免疫细胞化学显示，在两种温度下，>95%的细胞表达大T抗原。这些细胞在34°C下呈指数增殖，倍增时间为102天，但在40°C下不可逆地停止分裂。然而，当细胞在较高温度下维持6天时，细胞体积增加>2倍。该克隆在两种温度下均表达α1型（I）前胶原mRNA并分泌I型前胶原C肽，尽管在40°C下水平略有升高。细胞系在34°C下表达碱性磷酸酶活性，并且该酶的基础水平在40°C下增加2至6倍。碱性磷酸酶活性在两种温度下均被1α，25-二羟基维生素D3（维生素D3）诱导4 - 8倍，但转化生长因子-β1（TGF-β1）在40°C下抑制酶表达>90%。当克隆维持在34°C时，维生素D3还诱导骨钙素分泌增加10倍，并且这种诱导在40°C时增强> 8倍。甲状旁腺激素和毛喉素刺激细胞在34°C下产生4至6倍的细胞内环AMP（cAMP），这种刺激在40°C下增强2至4倍。相比之下，前列腺素E2仅在细胞维持在34°C时刺激cAMP增加7至8倍。该细胞系在34°C下分泌TGF-β1和白细胞介素-6（IL-6），但在40°C下仅IL-6的基础分泌增加70%。最后，茜素红-S组织化学染色表明，这些细胞在两种温度下都产生矿化结节。总之，本研究的结果表明HOB-02-C1细胞具有成熟的成骨细胞表型。因此，这种新的细胞系和其他以类似方式获得的细胞应该是有价值的体外工具，成人成骨细胞生物学的细胞，生物化学和分子研究。
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°C but revert to a normal phenotype at the nonpermissive temperature of 40°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°C with a doubling time of ˜2 days but irreversibly stop dividing at 40°C. However, cell volume increases >2‐fold when the cells are maintained for 6 days at the higher temperature. This clone expresses α1 type (I) procollagen mRNA and secretes type I procollagen C‐peptide at both temperatures, although the levels were slightly elevated at 40°C. The cell line expresses alkaline phosphatase activity at 34°C, and the basal level of this enzyme increases 2‐ to 6‐fold at 40°C. Alkaline phosphatase activity is induced 4‐ to 8‐fold by 1α,25‐dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor‐β1 (TGF‐β1) suppresses enzyme expression >90% at 40°C. Vitamin D3 also induces a 10‐fold increase in osteocalcin secretion when the clone is maintained at 34°C, and this induction is enhanced > 8‐fold at 40°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°C, and this stimulation is enhanced 2‐ to 4‐fold at 40°C. In contrast, prostaglandin E2 stimulates a 7‐ to 8‐fold increase in cAMP only when the cells are maintained at 34°C. This cell line secretes TGF‐β1 and interleukin‐6 (IL‐6) at 34°C, but only the basal secretion of IL‐6 increases 70% at 40°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.
影响因子：
4.2
作者：
道脇幸博
通讯作者：
道脇幸博
影响因子：
11.2
作者：
T. Fujiwara;E. A. Grimm;T. Mukhopadhyay;Wei-wei Zhang;Laurie B. Owen-Schaub;J. Roth
通讯作者：
T. Fujiwara;E. A. Grimm;T. Mukhopadhyay;Wei-wei Zhang;Laurie B. Owen-Schaub;J. Roth
增强破骨细胞样细胞形成的人骨髓基质细胞系的开发和表征。
影响因子：
4.8
作者：
Takahashi,S;Reddy,SV;Dallas,M;Devlin,R;Chou,JY;Roodman,GD
通讯作者：
Roodman,GD