IMMORTALIZATION OF HUMAN SALIVARY EPITHELIAL CELL LINESS

唾液腺由相对异质的细胞群组成， 显示特定标记的浆液、粘液、肌上皮和导管细胞 分化功能。 然而，人们对它知之甚少。 生物化学和分子机制， 是协调和调节的。 此类研究 由于缺乏同质细胞群的可用性而受到阻碍， 表现出唾液特异性分化。 因此，本项目的主要目标 项目是建立永生化的浆液性，粘液性，导管性 和肌上皮细胞，从人类唾液腺，使用致癌 病毒，用病毒DNA转染，以及通过改变培养条件。 具体地说，我们计划使用涉及分离细胞的程序， 包含完整的转化早期区域的亚基因组片段 AD 12-SV/40杂合病毒的突变体、野生型SV 40病毒的突变体和 SV 40的tsA 255温度敏感突变体，以及含有该突变体的质粒载体 致癌基因如v-Ha-ras。 将病毒DNA序列整合到 宿主细胞染色体和病毒基因的表达将通过 Southern印迹和免疫细胞化学分析。 如果这些 程序不成功，我们将测试涉及感染的程序 用SV 40-病毒和Ad 12-SV 40杂合病毒的细胞培养物。 的 细胞将通过超微结构参数表征; 细胞特异性标志物即淀粉酶的免疫细胞化学定位， 乳铁蛋白、分泌组分、溶菌酶和粘蛋白分泌;以及通过 检测细胞特异性mRNA。 此外，cDNA文库将被 制备并用于鉴定在特定唾液腺中表达的基因 细胞 这些基因将通过DNA测序及其 通过PCR和北方印迹证实在特定细胞中的表达 分析。 人涎腺上皮细胞的特性 与研究界的联系将有助于对分子生物学的研究。 调节各种细胞类型分泌的机制。

Salivary glands consist of relatively heterogenous cell populations of serous, mucous, myoepithelial, and duct cells that exhibit specific markers of differentiation functions. Little however, is known about the biochemical and molecular mechanisms by which the production and secretion of the specialized molecules are coordinated and regulated. Such studies are hindered by a lack of availability of homogeneous cell populations that exhibit salivary specific differentiation. Thus the main objective of this project is to establish immortalized populations of serous, mucous, ductal and myoepithelial cells, from the human salivary glands,using oncogenic viruses, transfection with viral DNA, and by modifying culture conditions. Specifically, we plan to use procedures involving transfecting cells with subgenomic segments containing the intact transforming early regions of mutant of AD12-SV/40 hybrid virus, of wild-type SV40 virus, and of the tsA255 temperature sensitive mutant of SV40, and plasmid vectors containing oncogenes such as the v-Ha-ras. Integration of viral DNA sequences into host cell chromosomes and the expression of viral genes will be examined by Southern blot and immunocytochemical analyses, respectively. If these procedures are not successful, we will test procedures involving infection of the cell cultures with SV40- virus and Ad12-SV40 hybrid virus. The cells will be characterized by ultrastructural parameters; immunocytochemical localization of cell specific markers i.e. amylase, lactoferrin, secretory component, lysozyme, and mucin secretion; and by detecting cell specific mRNAs. Additionally cDNA libraries will be prepared and used in identifying genes expressed in specific salivary gland cells. The genes will be characterized by DNA sequencing and their expression in specific cells will be confirmed by PCR and Northern blot analyses. The availability of well characterized human salivary gland epithelial cell lines to the research community would facilitate studies on the molecular mechanisms that regulate secretions of the various cell types.