Immortalization of normal human salivary gland cells with duct-, myoepithelial-, acinar-, or squamous phenotype by transfection with SV40 ori- mutant deoxyribonucleic acid.
Immortalization of normal human salivary gland cells with duct-, myoepithelial-, acinar-, or squamous phenotype by transfection with SV40 ori- mutant deoxyribonucleic acid.
复制标题
通过用 SV40 ori 突变体脱氧核糖核酸转染，使具有导管、肌上皮、腺泡或鳞状表型的正常人唾液腺细胞永生化。
DOI：
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发表时间：
1993
期刊：
影响因子：
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通讯作者：
Mitsunobu Sato
中科院分区：
文献类型：
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作者：
Masayuki Azuma;T. Tamatani;Y. Kasai;Mitsunobu Sato
背景
主要根据形态和功能差异，在人类唾液腺中识别出 4 种不同类型的细胞。为了更好地了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，我们尝试在体外系统中建立正常的人类唾液腺细胞。
实验设计
原代培养的人唾液腺细胞用 SV40 的起源缺陷突变体 DNA 转染。转染2至3周后，出现由小型致密细胞组成的缓慢扩张的集落，而模拟转染的细胞不再生长并最终进入危机，随后细胞死亡。
结果
使用有限稀释技术，我们从单个菌落中分离出 4 个具有不同形态的细胞克隆。对塑料培养皿中培养的细胞进行形态学观察，精确揭示唾液腺组成细胞的特征；即，显示立方形（NS-SV-DC）、纺锤形（NS-SV-MC）和扁平形（NS-SV-SC）形态的三个细胞克隆分别与导管、肌上皮和鳞状细胞相似。剩余的细胞克隆呈多边形，具有大量分泌颗粒（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SV40 DNA的整合和表达。所有细胞克隆均未发现软琼脂中的贴壁依赖性生长和裸鼠的致瘤性。
结论
这些结果表明，在体外系统中完成了具有导管、肌上皮、腺泡或鳞状表型的细胞克隆的建立，并且基于软琼脂中的集落形成能力和裸鼠中的致瘤性的评估，这些细胞克隆被认为是非肿瘤性的。
BACKGROUND
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.
EXPERIMENTAL DESIGN
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.
RESULTS
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.
CONCLUSIONS
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.