Azuma M, Tamatani T, Kasai Y, Sato M
Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry.
Lab Invest. 1993 Jul;69(1):24-42.
Based on morphologic and functional differences mainly, 4 distinct types of cells are recognized in human salivary glands. For a better understanding of cellular proliferation and differentiation of human salivary glands as well as carcinogenesis of salivary gland neoplasms, we attempted to establish normal human salivary gland cells in an in vitro system.
Primary cultured human salivary gland cells were transfected with origin-defective mutant DNA of SV40. After 2 to 3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, whereas mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death.
Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphologic observation of cells cultured on plastic dishes precisely revealed the characteristics of constituent cells of salivary glands; i.e., three cell clones showing cuboidal (NS-SV-DC), spindle (NS-SV-MC), and flattened (NS-SV-SC) morphology were similar to duct, myoepithelial, and squamous cells, respectively. A remaining cell clone showing polygonal in shape with numerous secretory granules (NS-SV-AC) resembled acinar cells. Characterization of cell clones by the ultrastructural examination and the search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Integration and expression of SV40 DNA were confirmed by Southern blot and indirect immunofluorescence staining. Anchorage-independent growth in soft-agar and tumorigenicity in nude mice were not recognized in all cell clones.
These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft-agar and tumorigenicity in nude mice, these cell clones are considered to be non-neoplastic.
主要基于形态学和功能差异，人类唾液腺中可识别出4种不同类型的细胞。为了更好地理解人类唾液腺的细胞增殖和分化以及唾液腺肿瘤的致癌作用，我们试图在体外系统中建立正常人唾液腺细胞。
用SV40的原点缺陷型突变DNA转染原代培养的人唾液腺细胞。转染2至3周后，出现了由小而紧密的细胞组成的缓慢扩增的集落，而 mock 转染的细胞不再生长，最终进入危机期，随后细胞死亡。
使用有限稀释技术，我们从单个集落中分离出4个具有不同形态的细胞克隆。在塑料培养皿上培养的细胞的形态学观察精确地揭示了唾液腺组成细胞的特征；即，三个呈现立方形（NS-SV-DC）、纺锤形（NS-SV-MC）和平扁形（NS-SV-SC）形态的细胞克隆分别类似于导管细胞、肌上皮细胞和鳞状细胞。剩下的一个呈现多边形且有许多分泌颗粒的细胞克隆（NS-SV-AC）类似于腺泡细胞。通过超微结构检查和寻找特异性抗原对细胞克隆进行表征，结果表明NS-SV-DC、NS-SV-MC、NS-SV-AC和NS-SV-SC分别与导管细胞、肌上皮细胞、腺泡细胞和鳞状细胞相似。通过Southern印迹和间接免疫荧光染色证实了SV40 DNA的整合和表达。在所有细胞克隆中均未发现软琼脂中不依赖贴壁生长和裸鼠致瘤性。
这些结果表明，在体外系统中成功建立了具有导管、肌上皮、腺泡或鳞状表型的细胞克隆，并且基于软琼脂中集落形成能力和裸鼠致瘤性的评估，这些细胞克隆被认为是非肿瘤性的。
