Lustig R H, Hua P, Yu W, Ahmad F J, Baas P W
Department of Pediatrics, University of Wisconsin, Madison 53792.
J Neurosci. 1994 Jun;14(6):3945-57. doi: 10.1523/JNEUROSCI.14-06-03945.1994.
Estrogen alters neurite outgrowth, neuritic spine development, and synaptogenesis in estrogen-responsive areas of the rat brain. However, examination of the specific effects of estrogen on neurons in vivo has been difficult. An in vitro model for the effects of estrogen on neurons was developed, using the PC12 rat pheochromocytoma cell line. Wild-type cells (PC12-WT) were stably transfected either with an expression vector coding for the full-length cDNA for the human estrogen receptor (hER), or with a control vector. Resultant clones were isolated, screened for incorporation of vector and expression of ER mRNA and protein, and analyzed for morphologic responses to estrogen. PC12-WT, NEO9 (ER-negative), and SER8 (ER-positive) cells exposed to 100 ng/ml NGF exhibited dose-responsive neurite outgrowth within 2 d by light microscopy (LM). Coadministration of 10(-10) to 10(-9) M estradiol (E2) had minimal effects on neurite outgrowth, neuritic spine development, or interneuritic connections in NEO9 or PC12-WT cells, but in SER8 cells E2 led to additive and dose-dependent increases in neurite outgrowth, spine development, and interneuritic connectivity. Coincubation of SER8 cells with E2 and the antiestrogen ICI 164,384 negated estrogenic effects on spine development and interneuritic connectivity. At the electron microscopic (EM) level, intercellular abutments of NEO9 or PC12-WT cells contained few and rudimentary gap junctions, with no increase by E2. However, SER8 cells exhibited augmented basal frequencies of gap junctions that increased with E2 incubation. Microinjection of Lucifer yellow into PC12-WT and NEO9 cells demonstrated low frequencies of dye coupling and no change with E2, but SER8 cells demonstrated increased dye-coupling frequency with E2 coincubation. The results suggest that SER8 cells recapitulate estrogen effects on neurons in vivo. Estrogen appears to induce an inherent neural morphologic program in estrogen receptor (ER)-containing cells. These three cell lines provide a unique in vitro system for studying mechanisms of estrogen-neuron interactions.
雌激素会改变大鼠脑中雌激素反应区域的神经突生长、神经棘发育和突触形成。然而，在体内研究雌激素对神经元的具体作用一直很困难。利用PC12大鼠嗜铬细胞瘤细胞系，建立了一个研究雌激素对神经元作用的体外模型。野生型细胞（PC12-WT）用编码人雌激素受体（hER）全长cDNA的表达载体或对照载体进行稳定转染。分离得到所得克隆，筛选载体整合情况以及ER mRNA和蛋白的表达情况，并分析其对雌激素的形态学反应。通过光学显微镜（LM）观察，暴露于100 ng/ml神经生长因子（NGF）的PC12-WT、NEO9（雌激素受体阴性）和SER8（雌激素受体阳性）细胞在2天内呈现出剂量依赖性神经突生长。联合给予10⁻¹⁰至10⁻⁹ M雌二醇（E2）对NEO9或PC12-WT细胞的神经突生长、神经棘发育或神经突间连接影响极小，但在SER8细胞中，E2导致神经突生长、棘发育和神经突间连接呈累加性和剂量依赖性增加。将SER8细胞与E2和抗雌激素ICI 164,384共同孵育可消除雌激素对棘发育和神经突间连接的影响。在电子显微镜（EM）水平，NEO9或PC12-WT细胞的细胞间连接很少且有基本的缝隙连接，E2未使其增加。然而，SER8细胞的缝隙连接基础频率增加，且随着E2孵育而升高。向PC12-WT和NEO9细胞中微量注射荧光黄显示染料偶联频率较低，E2处理后无变化，但SER8细胞与E2共同孵育后染料偶联频率增加。结果表明，SER8细胞重现了雌激素在体内对神经元的作用效果。雌激素似乎在含雌激素受体（ER）的细胞中诱导了一种内在的神经形态学程序。这三种细胞系为研究雌激素与神经元相互作用的机制提供了一个独特的体外系统。
