2024年2月16日发(作者：)

中国科学技术大学

博士学位论文

糖皮质激素对前额皮层促肾上腺

皮质激素释放因子的调控

作者姓名：

孟 庆 元

学科专业： 神经生物学

导师姓名： 周江宁 教授

完成时间： 二○一○年三月七日

University of Science and Technology of China

A dissertation for doctor’s degree

The regulatory effects of

glucocorticoid on the

prefrontal cortex

corticotropin-releasing factor

expression

Author’s Name： Qing-Yuan Meng

speciality： Neurobiology

Supervisor： -Ning Zhou

Finished time: March 7th, 2010

书脊

糖

皮

质

激

素

对

前

额

皮

层

促

肾

上

腺

皮

质

激

素

释

放

因

子

的

调

控

二

十

一

系

孟

庆

元

中国科学技术

大学

中国科学技术大学学位论文原创性和授权使用声明

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保密的学位论文在解密后也遵守此规定。

作者签名：_______________

年 月 日

摘 要

摘要

1. 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层中的调控

促肾上腺皮质激素释放激素（corticotropin releasing factor, CRF）被认为是应激反应的中枢驱动力，它在抑郁症的发病过程中起关键作用。CRF神经元被发现存在于前额皮层（PFC）的许多区域，该区域与情绪和认知的控制高度相关。然而，对于CRF在该区域的调控知之甚少。本实验中，我们的研究目的是确定急性束缚应激和糖皮质激素对于PFC CRF的调控作用，并阐明CRF在PFC中可能的作用。我们发现，急性束缚应激能够增加PFC CRF mRNA的表达，而糖皮质激素则降低其表达。在PFC糖皮质激素受体（GR）的表达与CRF神经元共存。此外，我们观察到在体内GR能被CRF启动子所招募。在原代培养的PFC神经元中，我们的研究还特别关注了CRF对CRF受体1（CRFR1）的作用。结果显示，CRF能够通过MEK-ERK1/2通路增加CRFR1的表达。总之，本研究可能有助于更好的理解CRF在PFC的功能。

2. 酸敏感离子通道3在大鼠下丘脑中的分布

酸敏感离子通道是质子门控电压非敏感阳离子通道，并且是表皮钠通道/退化因子超家族的成员。至今为止，在哺乳动物中已有6种酸敏感离子通道被识别并描述。在这些亚基之中，酸敏感离子通道3 (ASIC3)在啮齿动物中主要分布于外周神经系统，并认为与机械感受、化学感受及痛觉感受相关。但是，在大脑中我们对于ASIC3却知之甚少。我们因此运用逆转录聚合酶链式反应

(RT-PCR) 以及蛋白质免疫印迹 (Western Blot)的方法检测出ASIC3在大鼠的很多脑区都有表达，其中包括海马，杏仁核，尾壳核，前额皮层及下丘脑。我们还特别应用免疫组化的方法对ASIC3在大鼠下丘脑中的分布进行了研究。ASIC3免疫阳性分布很广泛并贯穿于整个下丘脑，在以下核团中的密度最高，它们是室旁核，视上核，视交叉上核，弓状核，下丘脑背内侧核，视前正中核，视前腹内侧核以及背侧结节乳头核。本研究有利于我们进一步了解ASIC3在中央神经系统的功能。

关键词：促肾上腺皮质激素释放因子，前额皮层，急性束缚应激，糖皮质激素，促肾上腺皮质激素释放因子受体1，酸敏感离子通道3，下丘脑

I

Abstract

Abstract

1. The regulation of Corticotropin Releasing Factor and Corticotropin

Releasing Factor Receptor 1 in rat prefrontal cortex

Corticotropin releasing factor (CRF) is considered as the central driving force in

the stress response and plays a key role in the pathogenesis of depression. CRF

neurons have been identified to locate in most regions of the prefrontal cortex (PFC),

a brain region that is highly associated with the control of emotion and cognition.

However, little is known on the regulation of CRF in this region. In this study, we

aimed to identify the regulatory effect of acute restraint stress and glucocorticoid on

PFC CRF and characterize the possible function of CRF in the PFC. We found that

acute restraint stress increased and glucocorticoid decreased PFC CRF mRNA

expression. The expression of glucocorticoid receptor (GR) was found to colocalize

with CRF neurons in the PFC. In addition, recruitment of GR by the CRF promoter

was observed in vivo. Specific attention was paid to the effect of CRF on CRF

receptor 1 (CRFR1) expression in primary PFC cultures. The results showed that

CRF increased CRFR1 expression through the MEK-ERK1/2 pathway. In summary,

this study may contribute to the better understanding of CRF functions in the PFC.

2. Distribution of Acid-sensing Ion Channel 3 in the Rat Hypothalamus

Acid-sensing ion channels (ASICs), the members of the epithelial sodium

channel/degenerin (ENaC/DEG) superfamily, are proton-gated voltage-insensitive

cation channels. Six ASIC subunits have been identified and characterized in the

mammalian nervous system so far. Of these subunits, ASIC3 has been shown to be

predominantly expressed in the peripheral nervous system of rodents and implicated

in mechnosensation, chemosensation and pain perception. Little is known on ASIC3

in the brain. We thus employed reverse transcription-polymerase chain reaction

(RT-PCR) and Western blot to examine the expression of ASIC3 in various rat brain

regions, including hippocampus, amygdala, caudate putamen, prefrontal cortex, and

hypothalamus. Specific attention was paid to the distribution of ASIC3 in the

hypothalamus of rats by using immunohistochemistry. ASIC3 immunoreactivity

showed a widespread pattern throughout the hypothalamus, with the highest density

in paraventricular nucleus, supraoptic nucleus, suprachiasmatic nucleus, arcuate

nucleus, dorsomedial nucleus, median preoptic nucleus, ventromedial preoptic

II

Abstract

nucleus, and dorsal tuberomammillary nucleus. This study may contribute to the

understanding of ASIC3 functions in the central nervous system.

Key words: CRF, PFC, acute restraint stress, glucocorticoid, CRFR1, ASIC3,

hypothalamus

III

目 录

目录

第一章 绪论„„„„„„„„„„„„„„„„„„„„„„1

1.1 引言„„„„„„„„„„„„„„„„„„„„„„„„„„„1

1.2 中枢神经系统CRF基因表达的生理调控„„„„„„„„„„„„2

1.2.1 CRF神经元的中枢分布和CRF基因的基础（非应激）表达„„„„„„„2

1.2.2应激刺激激活的中枢CRF系统和应激整合的神经环路„„„„„„„„„3

1.2.3糖皮质激素对CRF表达反馈调节„„„„„„„„„„„„„„„„„„5

1.3

CRF基因转录调控的分子机制 „„„„„„„„„„„„„„„„6

1.3.1 cAMP反应单元结合蛋白 „„„„„„„„„„„„„„„„„„„„„9

1.3.2活化蛋白1(Fos和Jun) „„„„„„„„„„„„„„„„„„„„„11

1.3.3皮质类固醇受体„„„„„„„„„„„„„„„„„„„„„„„„„12

1.3.4神经生长因子诱导的基因B(NGFI-B)„„„„„„„„„„„„„„„„15

1.3.5 CRF表达的其他调节因子„„„„„„„„„„„„„„„„„„„„„15

1.4 结论和今后的方向„„„„„„„„„„„„„„„„„„„„„17

参考文献„„„„„„„„„„„„„„„„„„„„„„„„„„„„18

第二章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层中的调控„„„„„„„„„„„„„„37

2.1 引言„„„„„„„„„„„„„„„„„„„„„„„„„„„37

2.2 材料与方法„„„„„„„„„„„„„„„„„„„„„„„„38

2.3 结果„„„„„„„„„„„„„„„„„„„„„„„„„„„42

2.4 讨论„„„„„„„„„„„„„„„„„„„„„„„„„„„50

参考文献„„„„„„„„„„„„„„„„„„„„„„„„„„„„54

第三章 酸敏感离子通道3在大鼠下丘脑中的分布 „„„„„„61

3.1 引言„„„„„„„„„„„„„„„„„„„„„„„„„„„61

3.2 材料与方法„„„„„„„„„„„„„„„„„„„„„„„„62

3.3 结果„„„„„„„„„„„„„„„„„„„„„„„„„„„64

3.4 讨论„„„„„„„„„„„„„„„„„„„„„„„„„„„70

IV

目 录

3.5 总结„„„„„„„„„„„„„„„„„„„„„„„„„„„72

参考文献„„„„„„„„„„„„„„„„„„„„„„„„„„„„73

第四章 讨论与总结„„„„„„„„„„„„„„„„„„„77

4.1关于前额皮层CRF调控及功能的讨论 „„„„„„„„„„„„„77

4.2关于ASIC3在下丘脑分布及功能的讨论 „„„„„„„„„„„„78

附录„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„80

攻读学位期间发表的学术论文与取得的其他研究成果„„„„„„„„„81

致谢„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„82

V

第1章 绪论

第一章 绪论

1.1 引言

动物必须不断地适应威胁体内平衡的内在及环境变化。稳态应变，一个首先由Sterling和Eyer提出的术语，指的是通过变化来保持稳定性(Sterling

and Eyer, 1981)。后来McEwen和同事把这个概念用于机体对应激因素的反应(McEwen and Stellar, 1993)。脊椎动物应激反应包括对内分泌，行为，自主神经，免疫和代谢的调整，使动物能够适应对机体平衡的挑战(Herman et al.,

1996; Speert and Seasholtz, 2001)。对应激因素的理解包括多种神经通路，他们最终汇聚作用于哺乳动物中定位在下丘脑室旁核（PVN）的肽能神经元，或者是非哺乳类的视前区（POA）。这些神经元产生促肾上腺皮质激素释放激素（CRF），CRF是对应激因素反应时，激活下丘脑－垂体－肾上腺／肾间（HPA／HPI）轴主要的神经荷尔蒙。CRF除了具有促垂体的功能，它还协调应激反应的很多方面，例如行为和自主神经的调整(Aguilera, 1998; Ziegler and Herman,

2002)。在非哺乳类物种中，CRF和相关肽是潜在的促甲状腺素（TSH）释放因子，它们通过激活甲状腺轴调节关键的生活历史变迁，例如两栖类的变态发育(Denver et al., 2002)。

CRF是一个41个氨基酸的多肽，首先在绵羊下丘脑中分离出来，根据其通过垂体前叶对促肾上腺皮质释放的促进作用而命名(Vale et al., 1981)。之后，在代表脊椎动物中每个纲的不同物种中都分离出了CRF，除了爬行动物(Lovejoy

and Balment, 1999)。CRF除了有促垂体的功能，它还广泛的分布在哺乳类及非哺乳类的大脑和脊髓(Cummings et al., 1983; Swanson et al., 1983; Zupanc

et al., 1999; Pepels et al., 2002; Lu et al., 2004; Richard et al., 2004;

Yao et al., 2004; Calle et al., 2005; Boorse and Denver, 2006)，在那里它作为一种神经递质/神经调质来协调行为和自主神经对应激的反应(Lovejoy and Balment, 1999; Orozco-Cabal et al., 2006)。CRF和相关肽被认为在饱感和摄食过程中起重要作用(Crespi and Denver, 2004; Crespi et

al., 2004; Mastorakos and Zapanti, 2004)，它们还会影响学习和记忆的巩固(Croiset et al., 2000; Gulpinar and Yegen, 2004; Fenoglio et al.,

2006)。CRF信号通路的组成部分有，CRF，urocortins，CRF受体和CRF结合蛋白，它们同样分布在许多外周组织，如肾上腺，脾，心脏，肺，肝，胸腺，胰1

第1章 绪论

脏，肠道，卵巢，精巢和胎盘（人类和高级灵长类）(Suda et al., 1984; Petrusz

et al., 1985; Muglia et al., 1994; Boorse and Denver, 2006)。CRF和相关肽可能影响到，如果不是全部，则是大部分的生理功能，包括神经，内分泌，血管，心血管，骨骼肌肉，免疫和生殖系统(Boorse and Denver, 2006; Boorse

et al., 2006)。CRF和HPA轴的紊乱被提示参与了几种人类病症的发病机制，如焦虑和抑郁，摄食障碍，炎症疾病，药物滥用和早产(Chrousos and Gold, 1992;

Majzoub et al., 1999; Tsigos and Chrousos, 2002)。

PVN/POA中促垂体神经元在脊椎动物脑中显示出最高水平的CRF表达，并在HPA/HPI轴的调控中扮演重要角色。在四足动物中，CRF神经元轴突投射到正中隆起，在那里释放神经荷尔蒙到垂体后叶循环并调控垂体前叶的功能。在硬骨鱼类，促垂体的CRF神经元投射到最接近的远端部，在那里它们释放其内容物到最接近的促肾上腺皮质细胞(Lovejoy and Balment, 1999)。这些视前区神经元已经关于调控CRH基因表达的因子受到极大重视(Doyon et al., 2003;

Yao et al., 2004)。在其它脑区，特别是边缘系统（杏仁核，终纹床核）和后脑（蓝斑）区域也含有CRF神经元，它们对应激刺激起反应，并可能被糖皮质激素所影响(Tsigos and Chrousos, 2002; Ziegler and Herman, 2002)。

1.2 中枢神经系统CRF基因表达的生理调控

1.2.1 CRF神经元的中枢分布和CRF基因的基础（非应激）表达

CRF在哺乳动物的整个大脑都有表达，包括边缘系统（海马，杏仁核，伏隔核和终纹床核），下丘脑，丘脑，大脑皮层，小脑和后脑(Cummings et al., 1983;

Swanson et al., 1983)。在非哺乳类物种中CRF分布方式与之相似，提示这种分布方式在脊椎动物进化过程中早已出现，并在强大的自然选择力量驱动下保留下来(Lovejoy and Balment, 1999; Yao et al., 2004)。该分布方式还提示基因调控序列在进化上是保守的，它决定了组织特异性的表达。

哺乳类下丘脑内主要合成CRF的地点是PVN的小细胞部。在非哺乳类物种中与之同源的区域是视前区（POA; preoptic nucleus）。四足动物中，这些神经元投射轴突到正中隆起外层的毛细血管，在那里CRF被释放进入门脉血液然后运送到垂体前叶(Aguilera, 1998)。垂体细胞在硬骨鱼类中直接被来自促垂体神经分泌神经元的投射所支配（也就是，那里没有正中隆起）(Lovejoy and

Balment, 1999)。哺乳动物PVNCRF的表达被若干脑区的刺激性输入所调控。CRF

mRNA和hnRNA在哺乳动物PVN的表达存在生理节律(Kwak et al., 1993; Watts

2

第1章 绪论

et al., 2004)。CRF基因表达的周期性被认为由视交叉上核（SCN）产生和维持(Szafarczyk et al., 1979)。来自海马的下行通路通过传导糖皮质激素的负反馈，在维持基础HPA活性的设定点中起重要作用(Jacobson and Sapolsky,

1991; Meijer and de Kloet, 1998)。

1.2.2 应激刺激激活的中枢CRF系统和应激整合的神经环路

在面临应激刺激时，HPA/HPI轴快速激活。四足动物中，CRF由正中隆起内改变的神经末稍在数秒内释放，运输至垂体前叶，在那里它激活CRF受体来增加促肾上腺皮质激素（ACTH）的生成。血浆中ACTH的增加导致由肾上腺皮质产生的GCs含量的增加，以及血浆中GC含量的增加。糖皮质激素在靶组织发挥各种效应为机体应对应激刺激动员能量。血浆GCs还在HPA轴的不同水平产生反馈来调节应激反应(Sapolsky et al., 2000; Tsigos and Chrousos, 2002;

Ziegler and Herman, 2002)。

PVN神经元树突末稍释放CRF垂体门脉血液是在应激反应中的重要事件，它介导了HPA/HPI轴的激活。当大鼠被暴露在不同的身体，生理或心理上的应激时（例如，足底电击，束缚，强迫游泳，乙醚刺激和内毒素刺激），CRF神经元已被证明其CRF mRNA的增加，以及一些情况下是hnRNA（CRF基因转录的标准）水平的增加(Herman et al., 1989; Imaki et al., 1991; Bartanusz et al.,

1993; Rivest et al., 1995; Kovacs and Sawchenko, 1996; Kovacs and

Sawchenko, 1996; Ma et al., 1997; Ma et al., 1997; Hsu et al., 1998; Chen

et al., 2001; Liu et al., 2001)。在Xenopus laevis中的实验结果显示，当暴露于一个摇晃/束缚应激刺激之后，其POA内CRF的免疫活性及hnRNA表现为迅速增加(Yao et al., 2004; Yao et al., 2007)。硬骨鱼类中的实验同样显示，应激刺激增加POA或下丘脑内的CRF mRNA水平(Doyon et al., 2003;

Huising et al., 2004; Doyon et al., 2005)及升高循环系统中CRF的浓度(Pepels et al., 2004; Flik et al., 2006)。综上所述，哺乳类，爪蟾和鱼类中的发现表明，应激刺激依赖的CRF基因的升高在脊椎动物进化中很早就已出现，暴露于应激刺激导致CRF基因转录的迅速增加。

下丘脑CRF神经元接受来自前脑边缘脑区，脑干自主神经中心和一些下丘脑本地核团的密集投射。与PVN之间没有密集的直接联系的脑区，如海马，杏仁核和蓝斑，可能通过中介机制间接的影响PVN神经元(Herman and Cullinan,

1997; Ziegler and Herman, 2002)。这些神经联系在基础状态和应激诱导的状态下，都在调控CRF基因活性中起重要作用。

大量及早基因的诱导(Honkaniemi et al., 1992; Cullinan et al., 1995)3

第1章 绪论

和CRF表达的增加(Kalin et al., 1994; Merali et al., 1998; Yao et al.,

2004)，提示边缘结构在对应激的反应中迅速激活。海马损伤显著增加大鼠内PVN CRF mRNA，以及Cynomolgus猴GC分泌超常(Herman et al., 1989; Sapolsky

et al., 1991)，表明该脑结构对PVN CRF表达具有抑制性作用。海马表现为对PVN有一个应激依赖的抑制性输入，因为刺激该区域降低HPA轴活性，而海马损伤增加ACTH和GC分泌对某些刺激的反应，但对其它的则不(Jacobson and

Sapolsky, 1991; Herman and Cullinan, 1997)。边缘系统被认为对心理的和多形态的刺激更为敏感，这些刺激需要更高次序的感觉加工过程(Herman and

Cullinan, 1997)。

杏仁核和终纹床核（BNST）等边缘结构与海马相比，似乎对PVN的CRF神经元有相反的影响。杏仁中央核在介导神经内分泌以及应激反应的其它方面起关键作用。电刺激该区域导致正中隆起CRF的分泌(Weidenfeld et al., 1997)。反之，非应激大鼠杏仁核的损伤降低PVN内CRF mRNA和正中隆起内CRF样的免疫活性(Beaulieu et al., 1989; Prewitt and Herman, 1994)，阻断CRF诱导的行为反应和HPA轴对声音和光刺激的反应(Liang et al., 1992)。注射CRF到大鼠杏仁核导致抑制性回避学习增加，探索行为降低(Liang and Lee, 1988)，而中央杏仁核CRF受体拮抗剂降低足底电击诱导的僵固行为(Swiergiel et al.,

1993)。因此，杏仁核环路可能对传导关于某些类型应激的刺激信号到PVN的CRF神经元十分重要(Gray et al., 1993; Herman and Cullinan, 1997)。

与杏仁核的损伤实验相似，非应激大鼠BNST的切除导致PVN内CRF mRNA的明显降低(Herman et al., 1994)。电刺激中央和喙端BNST导致血浆GC的增加(Dunn, 1987)，外侧BNST损伤也使恐惧诱导的ACTH及GC释放降低(Gray et

al., 1993)。此外，对于心理刺激的反应时，CRF mRNA在边缘系统增加（杏仁中央核和背外侧BNST），而下丘脑没有检测到升高(Makino et al., 1999)，提示边缘应激通路对于特定类型的应激刺激所起的反应不依赖于HPA轴的激活。

脑干对于自主神经系统对应激的反应十分重要。自主神经系统中的交感和副交感神经系统调节脉管系统，心脏，肠道和其他器官的骨骼肌和平滑肌，来与应激反应的其它方面相协调。脑干还介导觉醒，感觉加工和对生理刺激的反应(Brunson et al., 2001; Tsigos and Chrousos, 2002; Ziegler and Herman,

2002)。非应激大鼠中，半切除脑干下部（包括蓝斑）降低同侧PVN中CRF mRNA的表达(Kiss et al., 1996)，提示PVN基础CRF的表达处于脑干结构的兴奋性控制之下。切除蓝斑导致束缚应激后GC释放量的降低，提示从该区域对下丘脑神经分泌细胞存在刺激性的输入(Ziegler et al., 1999)。

CRF同样增加其自身以及CRF受体的表达，因此可能具有在一个组织中自4

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分泌或旁分泌的功能。脑室注射CRF及急性束缚应激能迅速诱导CRF和CRF受体mRNA在PVN的表达，提示CRF基因在应激反应中的自我调控(Luo et al., 1994;

Imaki et al., 1996)。用CRF受体1拮抗剂预处理能显著降低应激诱导的ACTH分泌和c-fos mRNA在PVN的表达(Imaki et al., 2001)，提示CRF的自调控有利于应激刺激依赖的CRF基因的激活以及/或者下丘脑神经元CRF的分泌。

除了CRF，其它神经肽如精氨酸加压素（AVP）和urocortins（CRF相关肽）也可能参与了应激对HPA/HPI轴的激活(Lovejoy and Balment, 1999;

Heinrichs and Koob, 2004; Suda et al., 2004)。

1.2.3 糖皮质激素对CRF表达反馈调节

糖皮质激素，HPA/HPI轴的最终效应者，在CRF表达和肾上腺/肾间活性调控过程起重要作用。应激刺激诱导产生了高浓度的循环系统GCs，它们对下丘脑CRF的合成和分泌，及垂体ACTH的产生和分泌有抑制效应。这种负反馈是终止应激反应使系统恢复稳态的重要机制(Makino et al., 2002)。在非应激的动物中，在PVN中CRF mRNA的水平与血浆中GC浓度呈负相关(Watts and Swanson,

1989; Kwak et al., 1992)。但是，肾上腺摘除的大鼠中仍维持CRF mRNA和hnRNA的生理节律，提示循环系统中的GCs不是CRF周期性表达所必需的(Kwak

et al., 1993; Watts et al., 2004)。

两种类型的细胞内GC受体表达在脑中：高亲和力的I型受体（也被称作盐皮质激素受体；MR）和低亲和力的II型受体（也被称作糖皮质激素受体；MR）(Funder, 1997)。GR分布在整个哺乳类脑中，其中在PVN和垂体前叶促肾上腺皮质激素细胞中表达水平最高。与之相对比的是，MR被发现在哺乳类海马中表达水平最高。因为它对糖皮质激素的高亲和力，表达在海马的MR被认为介导了GC在节律峰值时的负反馈作用，及维持HPA轴的基础活性(De Kloet et al.,

1998)。在急性应激之后，血浆中的GC浓度增加，低亲和力的受体GR可能扮演了重要角色，它介导了GC直接对PVN和垂体促肾上腺皮质激素细胞的反馈，通过负反馈终止HPA应激反应(De Kloet et al., 1998)。

GR抑制HPA轴的主导位点为PVN和垂体肾上腺皮质激素细胞。已经观察到激活的GR抑制下丘脑CRF基因的表达，并抑制垂体肾上腺皮质激素细胞中阿黑皮素原（POMC；包括ACTH在内的一系列多肽的前体蛋白）的表达(Dallman et al.,

1992)。GR还介导了GCs在边缘系统的反馈，包括海马，杏仁核，还有脑干神经元如蓝斑。这些神经核团与PVN的CRF神经元之间存在大量直接和间接的联系，GR在这些位点的激活参与调节HPA轴对急性及慢性应激反应的活性(De

Kloet et al., 1998)。另外，急性应激引起GR表达在PVN和垂体升高(Mamalaki

5

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et al., 1992)，而重复应激或持续给予外源性GCs则降低海马和杏仁核中的GR(Sapolsky et al., 1984)，为调节CRF表达和HPA活性提供了第二种机制。

大鼠中的研究表明，给予GC合成抑制剂甲吡酮导致PVN内的CRF hnRNA快速增加，接着是CRF mRNA的增加(Herman et al., 1992)。肾上腺摘除（ADX）也增加PVN内CRF mRNA，而高剂量的地塞米松对其有降低作用(Jingami et al.,

1985; Beyer et al., 1988)。这些结果提示在非应激状态时CRF基因的表达处于GCs持续的抑制效应之下。此外，在暴露于束缚或光刺激后大鼠PVN内CRF

hnRNA和mRNA的增加，ADX能增强而预处理地塞米松则抑制该效应(Imaki et al.,

1995; Feldman and Weidenfeld, 2002)。

然而在未经处理的动物中，生理浓度的GCs对起始和维持应激反应十分重要。事实上，ADX大鼠PVN中CRF hnRNA和mRNA的水平在应激刺激后降低(Tanimura and Watts, 1998)。补充低剂量GCs的ADX大鼠表现为血容量过少后PVN内CRF mRNA水平增加(Tanimura and Watts, 1998)。在另外一个大鼠中的研究，在肾上腺完整的动物中，CRF hnRNA和mRNA都增加，并且在持续性血容量过低的过程中保持增高(Tanimura and Watts, 2000)。但是，这些研究者发现，ADX动物血容量过低导致PVN CRF mRNA在早期增加，它会很快降低；对于ADX大鼠则没有观察到其PVN CRF hnRNA降低。此外，ADX大鼠的垂体后叶的CRF肽浓度降低(Plotsky and Sawchenko, 1987)，在GR表达减少的转基因小鼠与野生型相比，其刺激诱发内侧基底下丘脑分泌的CRF减少(Dijkstra et

al., 1998)。因此，血浆GCs明显对于PVN内CRF的表达施加了双向效应：高浓度循环的GCs（外源性的或应激过程中产生的）抑制PVN内CRF基因转录（负反馈效应），而低水平的GCs可能对于应激依赖的基因激活和分泌产生一个纵容效应，并保持应激反应起始阶段CRF的表达(Tanimura and Watts, 2001)。

MR参与负反馈调控HPA的证据来自于MR敲除小鼠的研究。与野生型相比，这些敲除鼠出现HPA轴活性的升高，表现为PVN mRNA水平的升高，血浆ACTH和皮质酮水平升高(Gass et al., 2001)。因此，GCs通过表达在边缘系统中的MR起作用，被认为对PVN的CRF基因产生一种“强直的”抑制作用，而GR可能介导了应激反应中对CRF“阶段性的”的调控(Gass et al., 2001)。

1.3 CRF基因转录调控的分子机制

CRF的表达在空间上，时间上和应激依赖性上在物种间的一致性提示，负责调控CRF基因的顺式反应元件出现在最早的脊椎动物中，因CRF在适应应激反应中的关键性作用，经过物种选择已经保持下来。 我们对脊椎动物CRF基因6

第1章 绪论

进行了基因组比较分析，发现了几个序列高相似性的区域（图1.1）。在编码区具有最高相似性的区域是在成熟肽段，而激素前体的结晶肽表现为保守性低很多。其内含子在脊椎动物的纲间事实上没有序列保守性，但是奇怪的是，在四足动物中，其3’非翻译区（3’UTR）出现几个高保守性的核酸序列的延伸。对CRF基因3’UTRs在鱼类和四足动物中进行更为细致的分析发现更低的序列保守性。四足动物3’UTR序列保守性的功能上的重要性尚不知晓，但是可能与转录或翻译所需的保守性元件相关。除了成熟的肽段，最近的启动子区域享有最高的序列相似性；在人类，大鼠和绵羊CRF基因，其上游330bp的序列的分析序列显示序列相似性达94%(Vamvakopoulos et al., 1990)，蟾蜍和哺乳类基因之间相似性达72%。

图1.1 脊椎动物CRF基因队列(按图中下行顺序排列)：河豚(Fugu rubripes)，鸡(Gallus

domesticus)，蛙(Xenopus tropicalis)，负鼠(Didelphis virginiana)，小鼠(Mus

musculus)，狗(Canis domesticus)和恒河猴(Macaca mulatta)基因组使用人作为基础序列。图中的峰代表了队列中基因组特定位点的同一性的百分比例。纵轴为减去了50%的同一性。队列是使用进化保守区域(ECR)浏览器完成的。具有最高保守性的区域是成熟的肽和最接近的启动子。神秘肽是激素原的一部分而且通过剪切可形成成熟肽。注意的是只有有限的5’端上游序列在当前的ECR浏览器(对alis和Fugu rubripes进行了更新)中是可用的(每个物种序列的长度是用每个柱状图下的灰色柱来表示)。

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图1.2 人(h)，黑猩猩(pt)，绵羊(sh)，小鼠(m)，大鼠(r)和非洲爪蟾(x；基因a和b)的CRF基因启动子区域的Clustal队列。相同的CRF基因序列用阴影表示。星号代表了每个基因预期的转录起始位点。推定的转录因子结合位点已经被标记出来了。NGFI-B，神经生长因子诱导基因B；ER，雌激素受体；Oct-1，八聚体结合转录因子-1；GR，糖皮质受体；AP1，活化蛋白1(Jun/Fos)；CREB，环化AMP反应元件结合蛋白；XFD2，爪蟾叉头区域因子2；Nkx 2-5，心脏特异性同源盒蛋白；AhR，芳香烃受体。

对邻近的CRF启动子的计算机分析发现几个可能的转录调节元件，包括两个TATA框，CAAT框，一个cAMP反应元件（CRE），几个AP-1蛋白（Fos/Jun）结合位点，几个半糖皮质反应元件（GRE），和半雌激素反应元件（ERE）(Vamvakopoulos et al., 1990; Vamvakopoulos and Chrousos, 1994)（图2）。邻近的启动子区域的主要顺式元件在脊椎动物CRF基因中高度保守(Yao et al.,

2007)（图1.2）。这些可能的调控元件中，其中一些人类和大鼠的CRF基因已经使用细胞转染和体外结合实验研究过。图1.3显示的就是对其中一些调控通路的一个系统图示。下面我们将对已被鉴定过的主要顺式反应元件的功能进行8

第1章 绪论

阐述，并就对其介导体内应激依赖的基因表达中的重要性提供证据的新结果加以讨论。

图1.3 示意图代表在最接近的启动子上与CRF基因转录调控有关的主要通路。PKA，蛋白激酶A；PKC，蛋白激酶C；GC，糖皮质激素；GR，糖皮质激素受体；GRE/AP1，组成性的糖皮质激素反应元件和AP1(Fos/Jun)结合位点；NGFI-B，神经生长因子诱导的基因B；NBRE，NGFI-B-反应元件；CRE，环化AMP反应元件；CREB，CRE结合蛋白；ICER，可诱导的cAMP早期抑制因子；JNK，Jun N末端激酶。TATA盒和起始位点也被标示出来。正、负调控分别用+、-标示。

1.3.1 cAMP反应单元结合蛋白

在许多细胞类型中已经证明cAMP依赖的蛋白激酶A(PKA)能激活CRF启动子活性：包括小鼠前垂体细胞AtT20(Van et al., 1990; Rosen et al., 1992;

Guardiola-Diaz et al., 1996; Malkoski et al., 1997; King et al., 2002)，大鼠嗜铬细胞瘤细胞PC-12(Seasholtz et al., 1988; Guardiola-Diaz et al.,

1994)，鸡巨噬细胞HD11(Van, 1993)，成神经细胞瘤SK-N-MC，绒毛膜癌细胞JAR(Spengler et al., 1992)，COS-7细胞(Vamvakopoulos and Chrousos, 1993)和人胎盘滋养层细胞(Cheng et al., 2000)。在原代胎鼠下丘脑细胞系中也已经证明了cAMP通路的激活能上调内源性CRF基因的表达(Emanuel et al.,

1990)。

cAMP反应元件结合蛋白(CREB)是基础亮氨酸拉链转录因子家族的成员。这个家族也包括cAMP反应元件调节蛋白(CREM)和活化转录因子(ATF-1)(Lonze

and Ginty, 2002)。这个家族的成员能通过他们的亮氨酸拉链区域形成同源或异源二聚体并与DNA结合。CREB的转录活性受到保守的丝氨酸残基磷酸化的调控，而且若干的胞内信号通路能诱导CREB的磷酸化，包括PKA，Ca2+，和分裂素激活蛋白激酶(MAPKs)(Shaywitz and Greenberg, 1999; Johannessen et al.,

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2004)。虽然磷酸化不能在体外改变CREB与DNA的结合(Richards et al., 1996;

Wu et al., 1998)，在体内实验中磷酸化已经显示能增加CREB DNA结合活性，而且不随总CREB蛋白水平的改变(Hatalski and Baram, 1997; Whitehead and

Carter, 1997; Wolfl et al., 1999; Hiroi et al., 2004)。CREB的磷酸化也诱导共活化因子CBP和p300与基因启动子的结合，这能通过内在的组蛋白乙酰转移酶活性和与RNA聚合酶II复合物的相互作用来促进基因的活性(Mayr

and Montminy, 2001; Johannessen et al., 2004)。一个高保守性的cAMP反应元件(CRE)TGACGTCA存在于人CRF基因启动子转录起始位点上游224bp的地方。这个元件有贡献于cAMP对异源启动子的响应，并且这个序列的突变能阻断8-bromo-cAMP或forskolin的诱导作用(Seasholtz et al., 1988; Spengler et

al., 1992)。以前的研究也已经显示这个在青蛙CRF基因启动子中的元件在体内或体外对cAMP通路介导的基因活性有响应(Yao et al., 2007)。

若干研究已经显示在大鼠中在应激的条件下CRF神经元中的CREB被快速的磷酸化，从而进一步导致在那些细胞中CRF表达的上升(Kovacs and Sawchenko,

1996; Chen et al., 2001; Bilang-Bleuel et al., 2002)。我们也显示青蛙受到摇晃应激在20分钟内增加POA区域CREB的磷酸化，而且，pCREB-ir与CRF-ir共定位(Yao et al., 2007)。虽然是间接的证据，这些结果支持这样的观点-在应激反应过程中CREB的激活能上调CRF基因的转录活性。

在大鼠PVN中显微注射8-bromo-cAMP能上调CRF mRNA的含量，然而预注射CREB的反义寡聚核苷酸能阻断由胰岛素诱导的低血糖症而导致的CRF mRNA的上调(Itoi et al., 1996)。我们最近报道在转染的蝌蚪脑中CRE位点的突变能阻断cAMP或应激依赖的青蛙CRF启动子活性(Yao et al., 2007)。这些结果强烈支持PKA/CREB通路能通过作用在CRF启动子的CRE位点而在体内环境中应激诱导的CRF表达起关键性作用。

在其他CREB家族成员中，最近的兴趣主要集中在可诱导的cAMP早期抑制因子(ICER)，它可能与应激依赖的CRF基因的活化密切相关。由仅有的DNA结合区域和CREM缺乏转录活性区域组成，ICER能与CREB和CREM形成二聚体并且能与公认的CRE位点结合，但是并不激活转录活性。但是，这个蛋白以作为cAMP诱导转录的有效的抑制因子起作用。ICER的表达能被其他CRE结合蛋白所诱导，例如CREB或CREM与ICER启动子的结合，并且它是CREB家族的仅有的成员，它能够被胞内浓度而不是磷酸化状态所调控。ICER的诱导能以一种反馈机制起作用，即下调由起始的CREB或CREM磷酸化诱导的活化作用(Sassone-Corsi, 1995; Mioduszewska et al., 2003)。在非应激的啮齿类动物中ICER免疫阳性被发现存在于下丘脑室旁核(Conti et al., 2004; Kell et

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al., 2004)。束缚应激能上调ICER mRNA及CREM对CRF启动子在下丘脑的招募，这可以下调应激后CRF mRNA的起始性诱导(Shepard et al., 2005)。抗抑郁处理也能增加ICER的表达，并且在小鼠中抗抑郁处理能降低应激诱导的血浆皮质酮水平(Conti et al., 2004)。另外，应激能在垂体和肾上腺都能诱导ICER mRNA的表达(Della Fazia et al., 1998; Mazzucchelli and Sassone-Corsi, 1999)，这表明ICER的上调可能是HPA轴调控的组成部分，而且这可能是应激激活的基因表达的反馈调控的共通机制。

1.3.2 活化蛋白1(Fos和Jun)

蛋白激酶C(PKC)通路通过转录因子Fos和Jun(原来称为活化蛋白1；AP-1)作用，被认为与CRF基因的转录激活有关。C-fos和c-jun属于即早基因(IEGs)家族，它们首先是基于能被生长因子快速诱导而被鉴别(McMahon and Monroe,

1992; Robertson, 1992)。C-fos的诱导(mRNA和/或Fos-ir)被广泛的用作神经元激活的标记物，而且还被用作在中央神经系统中确定应激反应回路(Ceccatelli et al., 1989; Hoffman et al., 1993; Senba and Ueyama, 1997;

Emmert and Herman, 1999)。在啮齿类动物中的研究显示在很多结构中c-fos

mRNA的增加对很广范围的应激因子响应，包括室旁核，中央和内侧杏仁核，终纹床核，海马和蓝斑(Ceccatelli et al., 1989; Imaki et al., 1992; Sawchenko

et al., 1996; Emmert and Herman, 1999; Watts and Sanchez-Watts, 2002)。与在啮齿类动物中的发现相一致，我们也发现青蛙暴露于摇晃应激其POA区Fos-ir明显增加，而且Fos-ir与CRF-ir共存(Yao et al., 2004)。这些结果显示AP-1蛋白可能在CRF基因对应激响应的调节起到了进化上保守的作用。

在COS-7或HD11细胞中转染人CRF启动子-报告载体，而且用佛波酯(TPA，能激活PKC通路)处理细胞，就能增加CRF启动子活性(Vamvakopoulos and

Chrousos, 1993; Van, 1993)。在人肝细胞瘤细胞系NPLC和胎鼠原代下丘脑细胞中检测mRNA水平，激活PKC通路也增加内源性CRF基因的表达(Emanuel et

al., 1990; Adler et al., 1992; Rosen et al., 1992)。TPA处理对CRF启动子活性的影响是依赖于细胞类型的，而且在转染的AtT20或PC-12细胞则没有观察到；而且在原代培养的大鼠杏仁核中TPA也不上调内源性的CRF基因的表达(Van et al., 1990; Rosen et al., 1992; Kasckow et al., 2003)。用TPA处理NPLC细胞引起的CRF mRNA增加需要de nove蛋白的合成(Adler et al.,

1992)。在这些细胞中，TPA也能通过3倍增加poly(A)尾端的长度增加CRF mRNA的大小，这可以潜在性的影响mRNA的稳定性或转录稳定性(Adler et al.,

1992)。

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若干AP-1结合位点存在于脊椎动物CRF基因的5’端区域(Vamvakopoulos

and Chrousos, 1994)（图1.2），虽然Fos/Jun在这些位点的结合还没有被识别。两个邻近的AP-1位点与3个公认的GRE位点相交迭，而且有结果显示AP-1蛋白和GR都能特异性的与人CRF启动子上的这些区域结合(Malkoski and Dorin,

1999)。我们也发现GR能与青蛙CRF基因的同源区域结合。此外，突变分析表明这个区域对于AP-1介导的对CRF启动子的活化和GR介导的对CRF启动子的抑制是非常重要的(Malkoski and Dorin, 1999)。组成的AP-1/GRE位点已经被发现于许多基因中，AP-1和GR对这些位点起相反的作用也已经被证明了(Diamond et al., 1990; Zhang et al., 1991; Miner and Yamamoto, 1992)。因此，Fos/Jun和GR(或MR)之间的拮抗或甚至是相互协作可能是一种普通的调节现象。这个假设仍旧需要在CRF基因调节的环境中检测。

尽管有发现显示在体内应激能诱导CRF神经元中c-fos的表达，而且在体外研究中PKC通路能激活CRF基因的转录，其他研究则没有证据显示这条通路的作用。Itoi和他的同事观察到在清醒的大鼠的PVN中显微注射TPA不影响PVN中CRF mRNA水平；相反的，侧脑室注射反义寡聚核苷酸直接拮抗c-fos或c-jun

mRNA不影响胰岛素诱导的低血糖症而导致的CRF基因的激活(Itoi et al.,

1996)。其他研究也证明在受到应激刺激之后室旁核c-fos mRNA的增加比CRF

hnRNA的增加有些迟滞(Kovacs and Sawchenko, 1996; Kovacs and Sawchenko,

1996)。此外，在大鼠中阻断蛋白质合成能阻止Fos的诱导表达，但是对CREB的磷酸化或CRF hnRNA的表达对急性应激的反应没有效果(Kovacs et al.,

1998)。这些发现得出了这样的结论，在应激反应中早期的CRF表达不依赖于Fos，至少在PVN中是这样(Kovacs and Sawchenko, 1996; Kovacs and Sawchenko,

1996; Kovacs et al., 1998)。作为替代的是，CREB的快速磷酸化是在应激反应的早期诱导CRF基因表达的主要原因。PKC通路的效应因子Fos/Jun可能在持续的或慢性应激中起到迟滞的CRF基因的调节作用。

1.3.3 皮质类固醇受体

糖皮质受体在基底及应激诱导的CRF基因表达方面起关键的作用。GC对于CRF表达的作用随着细胞类型、生理状态和激素的剂量密切相关。在啮齿类动物中的研究发现应用皮质酮能降低室旁核CRF mRNA水平，但是能增加杏仁中央核和终纹床核CRF mRNA水平(Makino et al., 1994; Makino et al., 1994)，这种情况表明GC对于CRF基因的表达有组织特异性的作用。用一种强力的糖皮质受体激动剂地塞米松(DEX)处理NPLC细胞能降低内源性的CRF mRNA。在转染的AtT20细胞中地塞米松能降低CRF基因启动子的基底活性(Adler et al.,

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1988; Rosen et al., 1992)。在AtT20细胞中，地塞米松也能明显的降低PKA通路依赖的CRF启动子活性，而且TPA也能在COS-7和NPLC细胞中诱导CRF启动子活性(Van et al., 1990; Rosen et al., 1992; Vamvakopoulos and Chrousos,

1993; Guardiola-Diaz et al., 1996; Malkoski et al., 1997; King et al.,

2002)。相反的是，在PC-12细胞中，DEX能提高PKA通路依赖的对CRF mRNA的诱导，而且在转染的人胎盘细胞能提高CRF启动子的活性(Guardiola-Diaz et

al., 1996; Cheng et al., 2000)；但是在原代培养的杏仁核神经元中，DEX对于基底的和forskolin诱导的CRF表达没有任何作用(Kasckow et al.,

1997)。

GC调控CRF基因表达的细胞靶点和分子机制还没有完全被理解。正如之前描述的，室旁核CRF表达的调控是直接的，这可能与CRF启动子中的GRE半位点有关，也可能是间接的，这则可能与包含海马和杏仁核的复杂的边缘系统回路相关(Tsigos and Chrousos, 2002; Ziegler and Herman, 2002)。糖皮质激素能在这些边缘结构调节CRF表达，但是调节特性则与PVN不同。GC对PVN和边缘结构CRF基因直接的调控在很大程度上需要糖皮质激素受体(GR)和盐皮质激素受体(MR)(Cole et al., 1995; Tronche et al., 1999; Gass et al., 2000)。然而，有证据显示GC对海马和室旁核的快速反应则并不依赖这些受体(Chen and

Qiu, 1999; Borski, 2000; Norman et al., 2004)。

GR对靶基因的直接作用或者是通过DNA结合依赖的方式或者是通过DNA非依赖的机制。人CRF基因的若干区域已经被发现存在GR(Guardiola-Diaz et al.,

1996)。在其中的一个区域中，AP-1/GRE位点的复合物已经被证明对GC抑制8-bromo-cAMP诱导的CRF启动子活性非常重要(Malkoski et al., 1997;

Malkoski and Dorin, 1999)。我们已经发现使用电泳迁移率检测技术(EMSA)GR能够与青蛙CRF启动子的同源区域结合。然而，现在还不清楚的是，在任何物种中GR对CRF直接调节作用是否是通过与这些GRE DNA结合位点相结合。

若干证据表明GR这种与DNA结合的功能对于特定基因的转录激活是非常关键的(Reichardt et al., 1998; Scott et al., 1998)。相反，GR关于AP-1或NGFI-B依赖的抑制作用可能不需要GR与特定基因直接的相互作用，而是依赖于蛋白质-蛋白质之间的相互作用来抑制转录活性。事实上，GR被发现直接在体外与若干转录因子相互作用，包括AP-1 (Fos/Jun)，NGFI-B，CREB和核因子-κB (NF-κB)(Diamond et al., 1990; Jonat et al., 1990; Yang-Yen et

al., 1990; Imai et al., 1993; De Bosscher et al., 1997; Martens et al.,

2005)，而且一个功能性的DNA结合区域不需要GR在胶原酶启动子抑制AP-1(Jonat et al., 1990; Schule et al., 1990; Yang-Yen et al., 1990; Heck

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et al., 1994)或在POMC启动子抑制NGFI-B(Martens et al., 2005)。在表达DNA结合缺陷GR突变体的转基因鼠中，GR和其他转录因子之间的相互作用并没有被损害(Reichardt et al., 1998)。

除了蛋白质-蛋白质直接作用的模型之外，一种竞争机制也被提出来了，它提出GR通过与一些有限量的核共激活因子竞争，例如CBP，p300和类固醇受体共激活因子(SRC)-1，来拮抗其他转录因子(Kamei et al., 1996; Aarnisalo et

al., 1998; Sheppard et al., 1998)。然而，其他的研究已经反对这种竞争模型，他们的研究显示GR对NF-κB 的抑制作用并不被胞内的共转录因子的浓度所限制(De Bosscher et al., 2000; De Bosscher et al., 2001)。

GR也通过间接的抑制c-Jun N端激酶(JNK)和MAPKs例如胞外调节激酶(Mamalaki et al.)和P38来干扰目的基因的转录(Caelles et al., 1997;

Swantek et al., 1997; Hirasawa et al., 1998; Lasa et al., 2001)。在培养的细胞中，DEX增加MAPK磷酸酶MKP-1的表达(一种MAPKs的抑制因子)(Kassel et al., 2001; Lasa et al., 2002)。

另一种GCs可能调节CRF基因表达的机制是通过mRNA的反转。在大鼠下丘脑中移植皮质酮能显著性的降低CRF mRNA的半衰期，这表明除了他们作为转录抑制因子的作用，GCs也可能通过降低CRF mRNA稳定性下调CRF的表达(Ma et

al., 2001)。

1.3.3.1 GC信号的非基因组机制

除了GC作用的基因组机制，非基因组作用是由G蛋白偶联的膜受体所介导的事实已经被证明了(Chen and Qiu, 1999; Borski, 2000; Norman et al.,

2004)。在垂体的水平上，大鼠静脉注射GC能在15分钟内抑制CRF诱导的ACTH分泌，而这个效果能够被GR的拮抗剂RU486所阻断(Hinz and Hirschelmann,

2000)。相似的灌流培养的垂体中GC快速抑制CRF诱导的ACTH分泌已经被观察到(Widmaier and Dallman, 1984)，以及在原代大鼠垂体细胞中DEX能快速抑制CRF诱导的cAMP积累和ACTH分泌，这个过程不能被蛋白质合成的抑制剂所阻断(Bilezikjian and Vale, 1983; Iwasaki et al., 1997)。在下丘脑的水平上，在大鼠下丘脑移植灌流培养中，DEX能快速抑制本底CRF的释放(Suda et

al., 1985)。

在海马中，皮质酮在小鼠CA1区能快速增加突触增强(Wiegert et al.,

2006)。既然海马神经元投射往PVN，而且被认为通过GABA能中继机制影响CRF神经元(Herman et al., 1995; Herman et al., 2002)，GC能够快速的通过调节海马输出调节PVN神经分泌细胞的活性。总之，这些研究能强调这种快速的，膜介导的GC对HPA轴反馈的潜力。

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1.3.4 神经生长因子诱导的基因B(NGFI-B)

神经生长因子诱导的基因B(也被称为Nur77)是另一种能被应激快速诱导的IEG。NGFI-B及其相关蛋白Nurr1是属于类固醇/甲状腺素受体超家族的孤儿受体(Hazel et al., 1988; Milbrandt, 1988)。大鼠室旁核中NGFI-B的表达能够快速的被足底刺激(Rivest and Rivier, 1994)或辣椒素(Honkaniemi et

al., 1994)所诱导，而且在癫痫发作以后在其他脑区也有这种诱导作用(Watson

and Milbrandt, 1989)。在这里提到的CRF增加的相同的脑区，脑区注射CRF也能在30分钟内能增加NGFI-B mRNA的表达(Parkes et al., 1993)。这些结果表明NGFI-B可能与应激依赖的下丘脑神经元的激活相关。

公认的NGFI-B结合位点(NGFI-B反应元件;NBRE)包含有9bp的序列AAAAGGTCA(Wilson et al., 1991)。大鼠CRF启动子中的NBRE与公认的NBRE有一个bp的差别(Wilson et al., 1991)，但是这个反应元件的功能性的差异却还不清楚。我们的比较基因组学分析结果揭示了非洲爪蟾CRFb基因启动子中一个完美的NBRE，与人，黑猩猩，小鼠的CRFa基因相应区域的NBRE只有一个bp的差异(Yao et al., 2007)。在大鼠垂体前叶细胞系AtT20中，用CRF或forskolin处理能快速增加NGFI-B二聚体与核DNA的结合活性(Maira et al.,

2003)。对AtT20细胞进行相似的处理能诱导NGFI-B的表达，而且与NGFI-B表达载体共转能够很强的诱导CRF和POMC启动子的刺激活性(Murphy and

Conneely, 1997; Kovalovsky et al., 2002)。用ACTH处理小鼠肾上腺皮质细胞系Y1能快速诱导NGFI-B mRNA的转录，而且重组NGFI-B的表达能明显以剂量依赖性的方式增加类固醇生成酶-21-羟化酶基因的启动子活性(Wilson et

al., 1993)。另外，已经有显示说明NGFI-B能够拮抗GR的反应(Philips et al.,

1997; Okabe et al., 1998; Martens et al., 2005)。总的来说，NGFI-B在下丘脑能介导应激依赖的脊椎动物CRF基因的调控，在垂体皮质腺介导POMC基因的调控，在肾上腺介导类固醇生成酶的调控，而且在多水平上能调控HPA/HPI轴的活性(Murphy and Conneely, 1997)。

1.3.5 CRF表达的其他调节因子

其他内分泌因子，特别是性腺类固醇，已经被证明与CRF系统相互作用从而调节应激反应。例如，在CRF启动子中已经鉴别出了公认的雌激素反应元件，它们能够与雌激素受体(ER)结合并且能够在CV-1细胞中介导雌激素刺激效应(Vamvakopoulos and Chrousos, 1993)。另一方面，雌激素处理转染的人胎盘细胞能够下调8-bromo-cAMP刺激的CRF启动子活性(Ni et al., 2004)。对卵15

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巢摘除的猴子进行雌激素替代治疗能够上调下丘脑室旁核的CRF mRNA水平，这种效应能够被孕酮废止(Roy et al., 1999)。这些发现显示性激素对于CRF基因的调节是一种组织特异性的方式，而且这种直接的调节能够全部或部分的解释基础CRF表达的性二态性和应激反应的性别差异。抑制元素沉默转录因子/神经元限制性沉默因子(REST/NRSF)是一种锌指转录因子，被认为通过在非神经元细胞中与抑制元件-1/神经元限制性沉默元件(RE-1/NRSE)起到抑制神经元基因表达的作用(Kraner et al., 1992; Mori et al., 1992; Chong et al., 1995;

Schoenherr and Anderson, 1995)。之后的工作显示了一种更加复杂REST/NRSF基因表达调控方式，说明这种蛋白可能除了神经元限制性沉默因子以外还起着其他作用(Bessis et al., 1997; Kallunki et al., 1998; Palm et al., 1998)。在人，大鼠，小鼠，羊和光滑爪蟾的CRF基因的内含子中存在有高保守性的公认的RE-1/NRSE(Seth and Majzoub, 2001)。在鸡的CRF基因的内含子中也鉴别有公认的RE-1/NRSE位点（表1.1）。REST/NRSF结合到小鼠/大鼠CRF基因RE-1/NRSE位点并抑制其转录活性(Seth and Majzoub, 2001)。REST/NRSF可能在发育和组织特异性CRF基因表达过程中起作用，但是这种假设还没有检测过。

POU同源区域蛋白Brn-2也被证明与下丘脑CRF基因的调控有关。Brn-2对下丘脑CRF神经元的发育是必须的(Schonemann et al., 1995)。它在体外与CRF启动子相结合而且在异源系统中能增加CRF启动子的活性。但是现在还不清楚这个转录因子是否与应激依赖的CRF基因调节相关(Burbach, 2002)。

表1.1 抑制元件-1/神经元限制性沉默元件(RE-1/NRSE)的序列在人，小鼠，大鼠，绵羊，鸡和非洲爪蟾(CRFa)的CRF基因的内含子中被发现。高保守性的RE-1/NRSE位点存在于人，小鼠，大鼠，绵羊，鸡和非洲爪蟾(CRFa)的CRF基因的内含子中。在鸡的CRF基因的内含子中也确认了推定的RE-1/NRSE位点。这个位点的定位在不同物种之间存在很大的差异性。

细胞因子(白介素-1，-2和-6)，GABA和生物胺，例如乙酰胆碱、无羟色胺和去甲肾上腺素已经被证明无论在体内还是在体外都能调节CRF的表达(Itoi

et al., 1998; Pisarska et al., 2001)。然而，这些因子所诱导的胞内信号通路以及起始CRF调控的细胞进程还没有被阐明。

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1.4 结论和今后的方向

CRF不仅在HPA/HPI轴活性的调节过程中起到了关键的作用，而且还调节脊椎动物的协调行为以及自主方面的应激反应。中央神经系统中CRF的表达受到内分泌和神经信号的复杂调控。在分子水平上，多种胞内信号通路以及转录因子的相互配合是CRF基因转录调控的必要条件，但是我们对于其中的很多通路还知之甚少。例如，尽管在鉴别介导应激依赖转录的cis反应元件的过程中取得了进展，但是对于其基因调节序列以及组织特异性的或发育性基因的表达我们几乎还一无所知。比较基因组学为识别保守的cis调控反应元件提供了一个有力的工具，而cis反应元件对发育、组织特异性和应激依赖性的基因调控是非常重要的。今后的工作主要依赖比较生物学结合高生殖力的转基因脊椎动物模型，例如非洲爪蟾，应该能使我们更好的解释CRF表达的调控机制。

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第2章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层的调控

第二章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层中的调控

2.1 引言

促肾上腺皮质激素释放因子（CRF）是一个41-氨基酸的多肽，主要由下丘脑室旁核（PVN）产生，它在应激反应中起关键作用，并被认为是下丘脑-垂体-肾上腺（HPA）轴活性的中枢驱动力(Bissette, 1990; Swaab, 2003)。除了下丘脑，CRF神经元还分布在前额皮层（PFC）的大部分区域(Swanson et al.,

1983)，PFC与情感和认知的控制高度相关(Miller, 1999)。这些区域是调节ACTH-皮质酮介导的应激反应的环路组成部分，参与调节对应激的反应能力(Diorio et al., 1993)。前人的研究提示，在新皮层，CRF表现为作为一种神经调质，或者甚至可能为一种神经递质，参与介导觉醒，学习和焦虑的增加，并同时降低摄食和性行为及改变运动(Koob and Heinrichs, 1999; Smagin et

al., 2001; Roozendaal et al., 2002)。许多这些效应可以被脑室注射CRF所模拟(Sherman and Kalin, 1987; Takahashi et al., 1989; Dunn and Berridge,

1990; Koob and Heinrichs, 1999)。更为近期的另外一个研究表明，CRF和急性应激可以改变PFC锥体神经元中五羟色胺（5-HT）的功能(Tan et al., 2004)。

多种应激刺激都被报道能够增加PVN内CRF基因的表达，例如束缚应激，足底电击，血容量减少及血糖过低(Harbuz et al., 1994; Kalin et al., 1994;

Paulmyer-Lacroix et al., 1994; Imaki et al., 1995; Ma et al., 1997; Herman

et al., 1998; Hsu et al., 1998; Tanimura et al., 1998)。杏仁核CRF基因的表达在受到束缚应激后增加(Kalin et al., 1994; Hsu et al., 1998)。另外，大量研究提示糖皮质激素以非常复杂的方式对CRF基因表达进行调控(Schulkin et al., 1998; Schulkin et al., 2005)。简而言之，众所周知糖皮质激素在PVN负调控CRF基因的表达(Keller-Wood and Dallman, 1984;

Swanson and Simmons, 1989)。在另一方面，慢性或重复地外周注射皮质酮能够增加杏仁中央核（CeA）及终纹床核（BNST）CRF mRNA的表达(Makino et al.,

1994; Makino et al., 1994; Watts and Sanchez-Watts, 1995)。另外，肾上腺摘除（ADX）在CeA和PVN内对中枢CRF基因的表达有相反的效应，它能使CeA中CRF的表达降低，PVN内CRF表达升高(Palkovits et al., 1998; Viau et

al., 2001)。然而，应激和糖皮质激素是否能够调控PFC内CRF的表达迄今仍不清楚。

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第2章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层的调控

2.2 材料和方法

2.2.1 动物

实验使用成年Sprague-Dawley雄性大鼠，体重250-300g（安徽实验动物中心）。大鼠饲养在动物房中，控制12-h昼夜周期，8：照明开始，食物和水充足。本实验中动物的使用和保护依据由中国科学技术大学动物保护和使用委员会批准的国际性条例和协议来进行。实验过程中尽量减少动物的痛苦和减少使用动物的数量。

2.2.2 急性应激

在实验应激程序开始之前，每天在饲养室内触摸大鼠，持续7天。触摸包括拿起每只大鼠，一个很短的时间后并把它放回原笼。在触摸阶段结束都立即开始应激操作。大鼠被随机分成体重匹配的组，每组分别接受如下四种处理中的一种：触摸（n=5），束缚应激0.5h（n=6），束缚应激1h（n=6）或束缚应激3h（n=6）。在应激刺激之前，将大鼠从饲养室中运到一个邻近，照明明亮，安静的房间后，放入一个透明树脂玻璃的管（长23.5cm，直径7cm）内，尾巴伸出。管子的大小限制侧向和前/后的运动，但是不影响呼吸。所有的应激实验开始于早上8：30。三组接受急性束缚应激的大鼠分别在束缚开始后0.5h，1h，3h后断头处死。对照组大鼠直接从笼中取出并断头处死。所有的实验都在早上进行，大鼠处死的时间在9：00到11：30之间。

2.2.3 肾上腺摘除和皮质酮处理

成年雄性Sprague-Dawley大鼠用7%的水合氯醛（1ml/100g）麻醉，从腰部切口后，或是进行双侧肾上腺摘除（ADX）或是进行假手术（n=7）。ADX后，给予大鼠0.9%的生理盐水来替代水，从而保持其电解质的平衡。ADX组进而分成两组，一组接受安慰剂（含10%乙醇的生理盐水，sc，n=8），另一组接受皮质酮（10mg/kg，用含10%乙醇的生理盐水配制，sc，n=8），每日两次（8：和17：）。在第7天的早上，在接受最后一次注射之后，所有的大鼠立即断头处死。

2.2.4 Mifepristone处理

糖皮质激素受体拮抗剂（北京紫竹药业公司）（50mg/kg，sc）的注射，又38

第2章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层的调控

称为RU486，在我们之前的研究中已有使用(Wu et al., 2007)，在急性束缚应激之前20h和2h注射。RU486用生理盐水溶解。在实验过程中这些溶液即配即用，安慰剂为生理盐水。

2.2.5 前额皮层的分离和样本准备

所有的大鼠被断头处死，血样用含肝素钠抗凝剂的管子收集后在4℃离心，分离后的血浆储存在-80℃，检测使用大鼠皮质酮ELISA试剂盒（RapidBio Lab,

Calabasas, California）。迅速取出大脑，分离前额皮层组织块。分离大鼠PFC时，依据Paxinos和Watson的坐标(Paxinos and Watson, 1998)，从大脑的前部在bregma2.7的位置切下，从嗅裂位置去除其基底部分后即可收集。整个分离过程在每只大鼠断头后2分钟内完成。组织样品立即放入液氮，之后在-80℃保存直至下一步分析。冰冻组织使用Glas-Col’s可变速匀浆器，在冰Trizol试剂（Invitrogen, La Jolla, CA, USA）中匀浆，根据厂家说明书来抽提RNA和蛋白。

2.2.6 PFC细胞的原代培养

大鼠前额皮层培养按照之前所述进行(Wang et al., 2003)。简要地说，从18天大鼠胚胎中分离前额皮层，使用0.25%胰酶-EDTA（Invitrogen）孵育30分钟来分离细胞，然后用抛光后的吸头吹打以粉碎组织。细胞被种在预包被的6孔板（包被使用多聚赖氨酸，Sigma-Aldrich, St. Louis, MO）中用于mRNA分析，或者种在玻片上（置于6孔板中，多聚赖氨酸包被）用于免疫组化，种植密度为1×105每平方厘米，使用无酚红的DMEM（Sigma-Aldrich）加10%胎牛血清（Invitrogen）培养。一天后，培养基换成无酚红的Neurobasal培养基（1.5ml，Invitrogen）加2% B27无血清补充（Invitrogen），每3-4天换液一次。种植后第四天，用5-Fluoro-2’-deoxyuridine （20μg/ml, Sigma-Aldrich)处理以阻止非神经元细胞的细胞分裂，从而有助于稳定细胞数目。培养条件保持在37℃，含5% CO2的潮湿环境中。所有的处理在培养10天后进行。

为了研究糖皮质激素对于CRF表达的效应，四分之一的培养基替换为含不同的试剂的Neurobasal培养基：dexamethasone（Sigma-Aldrich），RU486（Sigma-Aldrich）和forskolin（Sigma-Aldrich），单独一种或是不同的组合。为了检测CRF对CRFR1表达的效应，加入CRF（Sigma-Aldrich）和U0126（Cell

Signaling Technology）。Dexmethasone和RU486先用乙醇溶解后，使用前稀释到Neurobasal培养基中。CRF用蒸馏水溶解。Forskolin和U0126用DMSO39

第2章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层的调控

（Sigma-Aldrich）溶解。各种培养基均有与其相对应的对照。

2.2.7 实时定量RT-PCR分析

大鼠前额皮层和原代前额皮层神经元的CRF和CRFR1 mRNA的表达使用实时定量RT-PCR进行分析。β-actin mRNA的表达作为内参进行检测。一般而言，使用1μg总RNA反转录得到cDNA。反转录的反应条件如下：100 pmol/L oligo-dT,

1 mmol/L dNTP, 200 units RNAsin和200 units MMLV逆转录酶 (Promega,

Madison, WI, USA)。反应条件为42℃ 1h。之后反应体系在72℃加热10min在冰上冷却。

实时定量PCR使用的如下所示。对于CRF（accession no. NM_031019），正向引物是正向引物是5'-AAAGGGGAAAGGCAAAGAAAAGG-3’ ，反向引物是

5’-TGCCTGAGAAACATCATCCACTGG-3’ ，反向引物是

5’-AAGGCAGACAGGGCGACAGAG-3’。 对于CRFR1（accession no. NM_030999），5’-TAATTGTAGGCGGCTGTCACCAAC-3’。 对于β-actin（accession no.

NM_031144），正向引物是5’-TTGCTGACAGGATGCAGAA-3’ ，反向引物是

5’-ACCAATCCACACAGAGTACTT-3’。所有的引物用Primer Premier 5.0设计。反转录的cDNA用于Q-PCR，反应使用SYBR Premix Ex Taq（Takara），30μl反应体系加入0.5μM引物。样品的扩增使用ABI Prism 7000（Applied

Biosystems, Foster City, CA, USA），条件为：50°C2分钟, 95°C10分钟变性，接下来40个循环，循环条件为95°C 15秒变性， 64°C1分钟退火及延伸。

使用△Ct法来确定目的基因mRNA的水平。首先，将目的基因mRNA的阈值循环数（Ct）的数值用内参β-actin的Ct值来校准：△Ct = Ct (目的基因) –

Ct (β-actin)。得到的数值进一步用对照组来校准：△△Ct =△Ct (实验组) –△Ct (对照组)。进而就得到了改变的倍数（2-△△Ct）。相对的靶基因的mRNA水平则表示多次分别的实验中计算出的平均改变的倍数。PCR反应进行两次并观察到相似的结果。

2.2.8 Western blot

Western blotting用于分析原代前额皮层神经元中ERK的活性。一般而言，每个样本使用20μg原代神经元提取物，在10%SDS聚丙烯酰胺凝胶分离后转移到PVDF膜（Millipore, Billerica, MA, USA）上。该膜使用含5%脱脂奶粉的PBS-tween缓冲液（0.01 M PBS加0.05% Tween 20）在37℃封闭1h，然后孵40

第2章 促肾上腺皮质激素释放因子和促肾上腺皮质激素释放因子受体1在大鼠前额皮层的调控

育一抗，一抗用含5%脱脂奶粉的PBS-tween缓冲液配制，室温孵育2h。Immunoblotting使用anti-p-ERK抗体（1:2000, Cell Signaling Technology）和anti-ERK抗体（1:2000, Cell Signaling Technology）。将膜清洗之后，孵育二抗，二抗为辣根过氧化物酶联接的羊抗兔IgG，条件为室温1h。清洗后进行显色，使用ECL Western blot系统（SuperSignal West Pico chemiluminescent

Substrate, Pierce, Rockford, IL, USA），反应按照厂家说明进行。Western

blotting进行两次并观察到相似结果。

Western blot同样用来检测在大鼠PFC中GR抗体（1:500, Santa Cruz

Biotechnology, Santa Cruz, CA, USA）的特异性，该抗体用于免疫荧光染色和染色质免疫共沉淀，方法如前所述。

2.2.9 免疫荧光染色

为了制备脑片，将成年Sprague-Dawley大鼠用7%水合氯醛（1ml/100g）麻醉后，通过升主动脉先灌注PBS，然后灌注4%0.1M磷酸盐缓冲液配制的多聚甲醛。灌注后，取出大脑，将其浸泡在同种固定剂中过夜。然后，将大脑用梯度乙醇脱水后用石蜡包埋。用Leica切片机（Leica RM 2135）切成6μm的连续冠状切片。切片在二甲苯中脱蜡并在梯度乙醇中进水。在用TBS（0.05 M Tris缓冲液，pH 7.4，含0.15 M NaCl）清洗后，切片用微波（700W）在0.05M柠檬酸盐缓冲液（pH 6.0）处理2×10分钟以修复抗原，然后室温冷却30分钟。

用TBS清洗切片后，将其孵育在0.5%trition X-100中30分钟以通透组织。切片用含5%羊血清的TBST在37℃封闭30分钟以降低非特异性结合，然后孵育一抗，一抗为小鼠源抗GR单克隆抗体（Santa Cruz Biotechnology）和兔源抗CRF多克隆抗体（Bachem, Torrance, CA, USA），用含5%羊血清的TBST配制，37℃孵育1h后4℃过夜。第二天将切片清洗后，孵育FITC-标记的羊抗小鼠抗体（1:200, Pierce, 227 Rockford, IL, USA）及生物素标记的抗兔抗体（1:200,

Vector, Burlingame, CA, USA），用含5%羊血清的TBST配制，37℃孵育1h。切片在清洗之后，孵育Cy3标记的链亲和素（1:500, Kirkegaard and Perry

Laboratories, Gaithersburg, MD, USA），37℃孵育30分钟。然后清洗切片并用甘油封片。

对于原代培养的神经元，大鼠的PFC细胞用4%PBS配制的多聚甲醛在室温固定15min。接下来的步骤如前所示。

荧光信号的检测使用共聚焦激光扫描倒置显微镜（LSM 510, Carl 236 Zeiss,

Maple Grove, MN, USA），配置40×/0.75 NA PlanApo物镜和使用Zeiss图像处理软件（LSM 5, Carl Zeiss）同步采集。图像的获取使用滤镜组合，适合特41