延髓雌激素受体基因启动子表达的性差异

doi:10.3969/j.issn.1673-5374.2013.02.007

摘要/Abstract

摘要：

雌激素受体α广泛分布在大鼠脑内，但对雌激素受体α基因启动子的组织或靶特异性的了解还不够深入。实验采用雌激素受体α基因启动子0/B控制下表达增强的绿色荧光蛋白的转基因大鼠，免疫组化结果发现在雌性和雄性转基因大鼠的最后区和孤束核均有绿色荧光蛋白标记细胞分布，且二者间有绿色荧光蛋白纤维形成的神经网络联系，雌雄大鼠最后区内绿色荧光蛋白细胞的数量明显多于雄性，呈现性差异，但孤束核内绿色荧光蛋白细胞没有性差异。在成年雌性大鼠，卵巢切除或17β雌二醇处理不改变最后区内绿色荧光蛋白细胞的性差异。提示雌激素在最后区的效应可能与启动子0/B控制下的雌激素受体α表达有关，成年期改变性激素环境的处理不影响最后区和孤束核内雌激素受体α的表达。

关键词: 神经再生, 基础实验, 雌激素受体基因启动子, 绿色荧光蛋白, 性差异, 最后区, 孤束核, 延髓, 雌激素, 大鼠, 基金资助文章, 图片文章

Abstract:

Estrogen receptor α is widely distributed in the rat brain, but the tissue- or target-specificity of the estrogen receptor α gene promoters remains unknown. In the present study, we used transgenic rats expressing enhanced green fluorescent protein under the control of the estrogen receptor α 0/B promoter to examine expression driven by this promoter in two significant nuclei that regulate cardiovascular activity, the area postrema and the nucleus tractus solitarius. Immunohistochemistry showed that enhanced green fluorescent protein-labeled cells were distributed in the area postrema and the nucleus tractus solitarius of both female and male transgenic rats, and a neural network of enhanced green fluorescent protein-positive fibers was seen between the area postrema and the nucleus tractus solitarius. The number of enhanced green fluorescent protein-labeled cells in the area postrema of female rats was significantly higher than in the males, but no significant difference was found in the number of enhanced green fluorescent protein-labeled cells in the nucleus tractus solitarius. The sex differences in the number of enhanced green fluorescent protein-labeled cells in the area postrema was not affected after ovariectomy or 17β-estradiol benzoate treatment in adult rats. Our results suggest that the effects of estrogen in the area postrema are related to the expression of estrogen receptor α under the control of the 0/B promoter, and changes in the sex hormone environment in the adult period do not affect estrogen receptor α expression in the area postrema or the nucleus tractus solitarius.

Key words: neural regeneration, basic research, estrogen receptor &alpha, promoter, green fluorescent protein, sex differences, area postrema, nucleus tractus solitarius, medulla, estrogen, rats, grant-supported paper, photographs-containing paper, neuroregeneration

. 延髓雌激素受体基因启动子表达的性差异[J]. 中国神经再生研究(英文版), 2013, 8(2): 149-155.

Chunxiao Zhang, Tomohiro Hamada. Sex differences in estrogen receptor promoter expression in the area postrema[J]. Neural Regeneration Research, 2013, 8(2): 149-155.

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引言

材料方法

Design

A randomized, controlled, animal study.

Time and setting

The experiments were performed at the Department of Physiology, Nippon Medical School, Japan from May to November 2007.

Materials

Transgenic rats expressing enhanced green fluorescent protein under the control of the estrogen receptor α 0/B promoter were provided by the Laboratory Animal Center of Nippon Medical School, Japan. The transgenic rats were generated as follows. Briefly, a plasmid containing the estrogen receptor α 0/B promoter sequence was amplified and SacI/AflII fragments of the plasmid were injected into the pronucleus of fertilized oocytes of Wistar rats and the rats were identified by southern blot analysis of tail DNA using ³²P-labeled probes^[21]. We used five adult male and 15 female transgenic rats, aged 8 weeks or 4 weeks (for manipulation of the gonadal steroid milieu, below). The rats were allowed free access to water and food and were housed under a 14 hour light/10 hour dark cycle at 23°C.

Methods

Manipulation of the gonadal steroid milieu

Female transgenic rats (4 weeks old, n = 5) were anesthetized, ovariectomized and subcutaneously implanted with a 5-mm silastic capsule (inner diameter, 1.47 mm; outer diameter, 1.96 mm; Dow Corning, Midland, Michigan, USA) containing 17β-estradiol benzoate (Sigma, St Louis, MO, USA)^[32]. These 17β-estradiol benzoate capsules have been reported to maintain the serum estradiol titer within the proestrus range^[32]. Female rats without 17β-estradiol benzoate treatment (n = 5) were also anesthetized and ovariectomized at 4 weeks of age.

Sampling method

The distribution patterns of the fluorescent cells were determined in the area postrema and the nucleus tractus solitarius of male and female transgenic rats at 8 weeks of age (n = 5). All animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.) and perfused through the heart with ice-cold 1% heparinized saline, followed by ice-cold 4% paraformaldehyde in 0.1 M PBS (pH 7.3). Brains were removed and postfixed in the same fixative at 4°C overnight and then transferred into 30% sucrose (Sigma) in 0.1 M PBS until they settled. According to the rat atlas by Paxinos and Watson^[33], coronal sections (30-μm thick) containing the area postrema and the nucleus tractus solitarius were cut on a freezing microtome. All sections were collected and stored at 4°C in 0.1 M PBS.

Detection of enhanced green fluorescent protein in the area postrema and the nucleus tractus solitarius by immunohistochemistry

All sections containing the area postrema and the nucleus tractus solitarius were subjected to immunohistochemistal staining with an antibody against enhanced green fluorescent protein to avoid the attenuation of enhanced green fluorescent protein fluorescence during observation. Briefly, free-floating sections were rinsed with 0.05 M PBS and then incubated in 3% H₂O₂/methanol to remove endogenous peroxidases. After further washes in PBS, sections were incubated with 1.5% normal goat serum (Molecular Probes, Eugene, OR, USA) in PBST (0.05 M PBS/0.05% TritonX-100) for 30 minutes at room temperature. Sections were then reacted with rabbit anti-green fluorescent protein polyclonal antibody (1:1 000; Molecular Probes) for 60 hours at 4°C, followed by visualization of the immunolabeling using an Alexa 488-conjugated anti-rabbit IgG (1:800; Molecular Probes). The sections were then mounted, dehydrated and coverslipped with Vecta Mount (Vector Laboratories, Burlingame, CA, USA). The number of enhanced green fluorescent protein-labeled and -immunoreactive cells in the area postrema and the nucleus tractus solitarius was determined under an Olympus BX50 fluorescence microscope (Tokyo, Japan) with a digital camera system (Nikon, Tokyo, Japan).

Statistical analysis

Data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA) and presented as mean ± SD. One-way analysis of variance was used to analyze the sex differences and the effects of hormonal manipulation on the nucleus tractus solitarius and the area postrema. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1内容介绍：
雌激素及其受体对调节生殖功能有重要作用，但它对中枢神经系统的功能也有明显影响，包括学习记忆、认知、细胞膜的兴奋性，并和一些女性更年期疾病有关。雌激素受体（包括雌激素受体α和雌激素受体β）在脑内的表达较广泛，为可视化雌激素α受体，日本医科大学生理学系生产了一种转基因大鼠，它的雌激素受体α基因启动子0/B上标记有增强的绿色荧光蛋白，即在荧光显微镜下可见脑内雌激素受体α阳性神经元呈现绿色荧光。2007年检查了该转基因动物前脑增强的绿色荧光蛋白的表达，发现前脑有许多脑区表达这种绿色荧光蛋白，但没有检查延髓的表达情况。

作为替代激素，17β-雌二醇被长期用于绝经后妇女以预防一些更年期疾病的发生，如发挥其心血管保护效应，预防高血压和卒中的发生。在许多实验性高血压的动物模型中，卵巢切除增加高血压的发生，而雌激素替代能减弱高血压的进程。近期的研究显示，血液中慢性输入低剂量血管紧张素II，可引起雄性小鼠和卵巢切除雌性小鼠的血压明显升高，但对正常的雌性小鼠无明显影响。阻断中枢雌激素受体或敲除雌激素受体基因，可增加血管紧张素II的高血压效应。这说明雌激素能降低血管紧张素II引起的高血压，其作用与脑内雌激素受体有关。

在中枢神经系统，室周器是循环激素与脑内进行信息交流的惟一结构。最后区是室周器的组成结构之一，是调控心血管活动的关键性核团。追踪研究发现，最后区发送密集的纤维投射到邻近的背内侧和背外侧孤束核，后者对调节自主神经活性尤其是心血管活动具有重要作用。在最后区和孤束核均有雌激素受体（包括雌激素受体α和雌激素受体β）的高表达。雌激素及其受体在心血管活动调控中枢的作用目前尚不十分清楚。在实验中检查了延髓最后区和孤束核——2个已知的调节血压的重要核团的增强的绿色荧光蛋白的表达，以了解雌激素及其α受体在血压调节中的作用。

2实验设计及构思：
主要调查延髓两个血压调节的重要核团——最后区和孤束核标记雌激素受体α的增强的绿色荧光蛋白的表达情况，是否有性差异（即性二态性），阐明高血压出现性差异的可能机制。
(1)正常雌性和雄性转基因大鼠最后区和孤束核的增强的绿色荧光蛋白表达情况。
(2)雌性转基因大鼠去除卵巢后最后区和孤束核的增强的绿色荧光蛋白表达情况。
(3)雌性转基因大鼠去除卵巢并补充雌激素后最后区和孤束核的增强的绿色荧光蛋白表达情况。
(4)确定转基因大鼠延髓增强的绿色荧光蛋白表达是否有性差异，及成年雌激素环境改变是否影响这种性差异。

3国内外相关研究介绍：
发表于Brain Res Mol Brain Res. 2005;139(1):42-51的文章发现免疫组化观察转基因大鼠前脑雌激素受体α基因启动子0/B上标记有增强的绿色荧光蛋白的分布，提示前脑很多区域有增强的绿色荧光蛋白表达，这种增强的绿色荧光蛋白分布与雌激素受体α分布相一致。Endocrinology. 2007;148(3):1144-1149中原位杂交显示雌性和雄性的视前区性二型核的生长抑素分布及围产期雌激素改变的影响，说明视前区性二型核生长抑素分布呈现两性差异且有雌激素依赖的特性，这种性差异是在围产期确定的。J Neurobiol, 1998,36,357-378免疫组化显示雌激素受体(雌激素受体α和雌激素受体β)在脑内的分布，显示脑内许多区域有雌激素受体的分布，为它们的中枢作用提供解剖学证据。Proc Natl Acad Sci USA. 1999;96(13):7538-7540免疫组化显示雄激素受体在内侧杏仁核内的分布及成年期性激素改变的影响，发现内侧杏仁核雄激素受体呈现性二态性且有雄激素依赖的特性，这种性差异是在成年期确定的。Endocrinology. 2010;151(4):1923-8使用雌激素受体α基因启动子0/B上标记有增强的绿色荧光蛋白的转基因大鼠，免疫组化显示其在性二型核的性差异及雌激素的影响，显示雌激素受体雌激素受体α基因启动子0/B在视前区性二型核性差异的形成中具有重要作用，围产期雌激素改变设计这种性差异形成。

4创新性：
在CNKI中，以“雌激素受体；性差异”为关键词，未搜到相关文献。在Pubmed中检索“etrogen receptor α; sex difference”，发现400篇相关文献；检索“sex difference; area postrema”，发现7篇相关文献；检索“estrogen receptor α; sex difference; area postrema”，未搜到相关文献；检索“estrogen receptor gene promoter; area postrema”，也未搜到相关文献。

实验使用转基因大鼠，它的雌激素受体α在基因启动子0/B控制下表达增强的绿色荧光蛋白，发现延髓最后区（血压调节的重要核团）的增强的绿色荧光蛋白细胞数量明显高于周围其他核团，且呈现明显的两性差异，这可能与雌激素抑制高血压的形成有关。