Galler Kerstin M, Schweikl Helmut, Thonemann Birger, D'Souza Rena N, Schmalz Gottfried
Department of Operative Dentistry and Periodontology, University of Regensburg, Germany.
Eur J Oral Sci. 2006 Apr;114(2):138-46. doi: 10.1111/j.1600-0722.2006.00327.x.
Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells.
培养中的原代细胞分裂能力有限，很快就会进入非增殖状态。这种细胞衰老限制了对源自人牙髓的细胞在细胞途径、基因调控、牙本质形成机制或对材料暴露的反应方面的研究。为了克服这个问题，建立了原代人牙髓来源的细胞，并将其用含有猴病毒40（SV40）大T抗原编码序列的质粒进行转染。这导致建立了几个显示寿命延长的细胞克隆。通过逆转录-聚合酶链反应（RT-PCR）和免疫组织化学验证了T抗原转录本和蛋白质的表达。原代人牙髓细胞培养至衰老（即传代至第7代），转染后的细胞在转染后可培养至第18代，此时发生了伴有大量细胞死亡的细胞危机。一个克隆逃脱了危机，并已在培养中维持了55周。进行实验以比较转染细胞与原代细胞的特征。分析了细胞形态和增殖情况，并通过RT-PCR和免疫组织化学检测了细胞特异性基因转录本和蛋白质（包括I型和III型胶原、碱性磷酸酶、骨唾液蛋白、骨钙素、牙本质涎磷蛋白和牙本质基质蛋白I）的表达。人牙髓来源细胞的转染产生了一个永生化细胞系，该细胞系保留了在原代细胞中观察到的许多表型特征。
