IMMORTALIZATION OF HUMAN SALIVARY EPITHELIAL CELL LINESS

唾液腺由相对不同的细胞群组成 浆液细胞、粘液细胞、肌上皮细胞和导管细胞表现出特定的标记 分化功能。然而，人们对此知之甚少 产生和分泌的生化和分子机制 的专门化分子是协调和调节的。这类研究 由于缺乏可利用的同质细胞群体， 表现为唾液特异性分化。因此，这一行动的主要目标是 项目是建立浆液性、粘液性、导管不朽的种群 和肌上皮细胞，来自人类唾液腺，使用致癌 病毒，用病毒DNA转染法，以及通过改变培养条件。 具体地说，我们计划使用涉及将细胞与 含有完整转化早期区域的亚基因组片段 AD12-SV/40杂合病毒、野生型SV40病毒和 SV40的温度敏感突变体tsA255和含有 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SV40病毒和AD12-SV40杂合病毒培养细胞。这个 细胞将通过超微结构参数进行表征； 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、分泌成分、溶菌酶和粘蛋白分泌； 检测细胞特异性mRNAs。此外，还将建立 制备并用于鉴定特定唾液腺表达的基因 细胞。这些基因将通过DNA测序和它们的 在特定细胞中的表达将通过聚合酶链式反应和Northern杂交得到证实 分析。 特性良好的人涎腺上皮细胞的可用性 与研究界的联系将促进对分子的研究 调节各种细胞类型的分泌的机制。

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.