Chen C L, Simmen R C, Chou J Y
Department of Large Animal Clinical Sciences, University of Florida, Gainesville 32610.
Chin J Physiol. 1991;34(1):65-80.
An ideal in vitro model for the study of endocrine functions would be one in which cells could propagate in culture and express their specialized functions. Most endocrine studies to date have relied on primary cell culture or on the use of tumor cell lines. This report describes the characterization of three endocrine cell lines immortalized by transfecting endocrine cells with a temperature-sensitive mutant SV40 virus. Rabbit endometrium (HRE-H9), human placenta (SPA209-10) and rat pituitary (RP) cells were immortalized by SV40 virus, a temperature-sensitive (ts) mutant in the A gene, which encodes the large tumor antigen that is required for the maintenance of transformation. The transformed phenotype of the SV40 tsA mutant-immortalized cell line can be reversed simply by a shift in temperature. At the permissive temperature (34 degrees C), all three types of cells exhibited a transformed phenotype, which is characterized by high cell density growth and by the overgrowth of nontransformed cell layers. However, at the non-permissive temperature (40 degrees C) these cells reverted to a non-transformed phenotype as demonstrated by a marked decrease in the overgrowth of nontransformed layers and by the expression of differentiated functions. At the non-permissive temperature (40 degrees C), the endometrial cell line was capable of synthesizing beta-endorphin, and it exhibited hormonally regulated expression of the transfected hybrid uteroferrin gene construct. The human placenta cell line was capable of secreting GnRH upon stimulation by cAMP, forskolin, theophyllin, PGE, catecholamine and Ca++ channel stimulators. Moreover, the rat pituitary cell line was capable of synthesizing and secreting growth hormone (GH) which was stimulated by GHRH and cAMP. The advantage of the temperature-sensitive cell lines is that a single cell line is the source of both the normal and transformed states; thus, studies are internally controlled. These results demonstrate that tsA mutants of SV40 virus are the best available agents for immortalizing mammalian endocrine cells that retain differentiated functions.
用于内分泌功能研究的理想体外模型应该是一种细胞能够在培养中增殖并表达其特定功能的模型。迄今为止，大多数内分泌研究依赖于原代细胞培养或肿瘤细胞系的使用。本报告描述了通过用温度敏感型突变SV40病毒转染内分泌细胞而永生化的三种内分泌细胞系的特性。兔子宫内膜（HRE-H9）、人胎盘（SPA209-10）和大鼠垂体（RP）细胞被SV40病毒永生化，该病毒是A基因中的温度敏感（ts）突变体，A基因编码维持转化所需的大肿瘤抗原。SV40 tsA突变体永生化细胞系的转化表型可通过温度变化简单逆转。在允许温度（34摄氏度）下，所有三种类型的细胞都表现出转化表型，其特征是高细胞密度生长和未转化细胞层的过度生长。然而，在非允许温度（40摄氏度）下，这些细胞恢复为未转化表型，这表现为未转化层的过度生长显著减少以及分化功能的表达。在非允许温度（40摄氏度）下，子宫内膜细胞系能够合成β-内啡肽，并且它表现出转染的杂合子宫铁蛋白基因构建体的激素调节表达。人胎盘细胞系在受到cAMP、福斯高林、茶碱、前列腺素E、儿茶酚胺和钙离子通道刺激剂刺激时能够分泌促性腺激素释放激素（GnRH）。此外，大鼠垂体细胞系能够合成和分泌生长激素（GH），其受到生长激素释放激素（GHRH）和cAMP的刺激。温度敏感细胞系的优点是单个细胞系是正常状态和转化状态的来源；因此，研究是内部对照的。这些结果表明，SV40病毒的tsA突变体是使保留分化功能的哺乳动物内分泌细胞永生化的最佳可用试剂。
