DEMONSTRATION OF STEROID-HORMONE RECEPTORS AND STEROID ACTION IN PRIMARY CULTURES OF RAT GLIAL-CELLS
DEMONSTRATION OF STEROID-HORMONE RECEPTORS AND STEROID ACTION IN PRIMARY CULTURES OF RAT GLIAL-CELLS
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DOI：
10.1016/0960-0760(92)90394-x
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发表时间：
1992-03-01
影响因子：
4.1
通讯作者：
BAULIEU, EE
中科院分区：
文献类型：
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作者：
JUNGTESTAS, I;RENOIR, M;BAULIEU, EE
以新生大鼠前脑为材料，建立了大鼠神经胶质细胞原代培养。通过对每种细胞类型进行特异性标记的间接免疫荧光染色证实，获得了少突胶质细胞和星形胶质细胞的混合群体。在原代培养3周后，在50 nM雌二醇存在或不存在的情况下，我们测量了胶质细胞的受体，我们已经鉴定了黄体酮、糖皮质激素、雌激素和雄激素受体（PR、GR、ER和AR），但只有PR能被雌激素诱导。结合研究和kc146抗PR单克隆抗体的免疫组织化学技术表明，雌性后代的神经胶质细胞比雄性后代的神经胶质细胞更明显受到雌激素的诱导。抗雌激素药他莫昔芬抑制雌激素诱导，但对PR浓度无影响。分别用[H-3]R5020、[H-3]地塞米松、[H-3] oh -他莫昔芬或[H-3]R1881标记细胞后，采用全细胞法测定PR、GR、ER和AR的特异性结合位点。PR和GR也通过超离心分析，在细胞暴露于激动剂后，两种受体都以9S形式从细胞质中回收，并从核高盐，含钨酸盐离子的部分中回收为4-6S形式。相反，当使用抗黄体酮和抗糖皮质激素RU486作为配体时，在该核片段中发现两种受体都具有非活化的8.5S受体复合物。在特异性抗体存在的情况下，进一步分析了GR的8.5S复合物，发现除了GR外，还存在热休克蛋白hsp90和59 kDa蛋白。在原代培养过程中，检测黄体酮(P)和雌二醇（E2）对胶质细胞增殖、形态和分化的影响。P抑制细胞生长，E2刺激细胞生长。这两种激素都能诱导少突胶质细胞和星形胶质细胞发生剧烈的形态变化，并增加少突胶质细胞中髓鞘碱性蛋白和星形胶质细胞中胶质原纤维酸性蛋白的合成。
Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [H-3]R5020, [H-3]dexamethasone, [H-3]OH-tamoxifen or [H-3]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4-6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presence of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found.During primary culture, the effects of progesterone (P) and estradiol (E2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E2. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in olgodendrocytes and of the glial fibrillary acidic protein in astrocytes.