Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector - Establishment of a novel cell line for the study of human nucleus pulposus cells
Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector - Establishment of a novel cell line for the study of human nucleus pulposus cells
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DOI：
10.1097/01.brs.0000131419.25265.23
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发表时间：
2004-07-15
期刊：
影响因子：
3
通讯作者：
Hotta, T
中科院分区：
文献类型：
--
作者：
Sakai, D;Mochida, J;Hotta, T
研究设计.使用重组猴病毒40（SV 40）腺病毒载体从人髓核细胞衍生的从头细胞系的建立和表征。评估人髓核细胞系获取的可行性，并评估所得结果的特征，以更好地了解人髓核细胞的性质。尽管最近在椎间盘细胞生物学研究方面取得了进展，但髓核细胞的基本性质，特别是在人类细胞系的背景下，仍然没有得到很好的理解。因此，对这些细胞进行广泛的分析是非常必要的。由于现有的人类细胞数量有限，建立永生细胞系将极大地促进资源供应。在发布知情同意书后，从一名因爆裂性骨折接受前路融合术的19岁男性的腰椎间盘中获取髓核组织样本。选择无明显损伤的样品，酶切原代培养，然后用重组SV 40腺病毒载体（Ad/SV 40）感染。将感染的细胞在培养物中维持超过40个群体倍增时间，之后将其视为永生化的。接着，确认T抗原的表达，将得到的永生化细胞系命名为Ad/SV 40感染-1（HNPSV-1）来源的人髓核细胞系。HNPSV-1细胞进行了表征，并在两种指定的培养条件下与其母细胞进行了比较：单层和三维。在不同的时间间隔进行形态学和免疫细胞化学分析。还进行了细胞增殖、DNA合成、蛋白聚糖合成、基因表达谱和核型分析。此外，将HNPSV-1细胞注射到兔椎间盘中以评估肿瘤发生的存在。重组SV 40腺病毒载体以相对高的效率感染髓核细胞（在感染复数100下为90%）。HNPSV-1细胞存活时间明显延长，连续培养5个月以上（60 - 100个细胞群）。尽管与其母细胞相比，细胞增殖和DNA合成显着增加，但所得细胞系表达出惊人相似的细胞形态和功能特征。HNPSV-1感染兔椎间盘后24周，未见明显的成瘤作用。用重组SV 40腺病毒载体成功构建了HNPSV-1。结果表明，人髓核细胞具有永生化的能力，并保持了原有的细胞特性。预期这些细胞将用于体外研究人髓核细胞的生物学性质。
Study Design. Establishment and characterization of a de novo cell line derived from human nucleus pulposus cells using a recombinant simian virus 40 (SV40) adenovirus vector.Objectives. To assess the feasibility of human nucleus pulposus cell line procurement and to evaluate the character of the resultant outcome to better understand the nature of human nucleus pulposus cells.Summary of Background Data. Despite recent advances in disc cell biologic research, the fundamental nature of nucleus pulposus cells, especially in the context of human cell lines, is still not well understood. Therefore, a broad-based analysis of these cells is of significant necessity. Because of the limited amount of existing human cells, establishment of an immortal cell line would greatly facilitate resource supply.Methods. After release of informed consent, tissue samples of nucleus pulposus were obtained from the lumbar intervertebral disc of a 19-year-old man undergoing anterior fusion for burst fracture. Samples with no apparent damage were selected and digested enzymatically for primary culture and then were infected with recombinant SV40 adenovirus vector (Ad/SV40). The infected cells were maintained in culture for more than 40 population doublings, after which they were considered immortalized. Next, confirmation of expression of T antigen was performed and resultant immortalized cell lines were designated and classified as human nucleus pulposus cell line derived from Ad/SV40 infection-1 (HNPSV-1). HNPSV-1 cells were characterized and compared with their mother cells under two designated culture conditions: monolayer and three-dimensional. Morphologic and immunocytochemical analyses were performed at various intervals. Cell proliferation, DNA synthesis, proteoglycan synthesis, gene expression profiling, and karyotypic analyses were also performed. Moreover, HNPSV-1 cells were injected into rabbit discs to assess the presence of tumorigenesis.Results. Recombinant SV40 adenovirus vector infected nucleus pulposus cells with relatively high efficiency (90% > at multiplicity of infection 100). HNPSV-1 demonstrated marked prolongation of cell life with continuous cell doublings for over 5 months ( 60 - 100 cell population doublings). Despite significant increase in cell proliferation and DNA synthesis when compared with its mother cells, resultant cell lines expressed strikingly similar cell morphology and functional characteristics. Atypical karyotypes were noted; however, no apparent tumorigenesis was seen in rabbit discs 24 weeks after injection of HNPSV-1.Conclusions. HNPSV-1 was successfully established using recombinant SV40 adenovirus vector. Results showed that human nucleus pulposus cells are capable of immortalization with maintenance of original cell characteristics. It is anticipated that these cells will be useful for in vitro studies of the biologic nature of human nucleus pulposus cells.