褪黑素在AD发病中的作用及其对钙超载和AD相关基因的影响
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摘要
阿尔茨海默氏病（Alzheimer's Disease，AD）是与老年人记忆丧失有关的
神经变性疾病，在老年人群中的发病率仅次于心脑血管疾病和癌症。AD
的病因是多方面的，最主要有：（1）神经细胞的损伤和老化；（2）遗传易感性，
其中研究得较清楚的几个与AD有关的基因有APP、PS-1、PS-2、ApoE
基因；（3）神经系统自身免疫。AD在发病中后期都出现氧化应激（oxidative
stress），这是AD重要和关键的病理生理过程。
褪黑素（melatonin，MT）的降低被认为是AD发生的一个重要原因，可
以解释AD病人海马去甲肾上腺素、5-HT的降低及单胺氧化酶活性的增加。
MT对AD的治疗作用成为近年的研究热点，这是因为MT具有以下特点：
1、容易通过血脑屏障。2、是目前所知最强效的自由基清除剂。3、是强效
亲脂性抗氧化剂。4、能阻止β-淀粉样蛋白的形成。
本实验采用原位杂交、RT-PCR、激光共聚焦、放免、电镜和Morris
水迷宫行为学检测等方法，观察了MT受体Mel 1a和Mel 1b在正常成年
大鼠中枢神经系统的分布及MT限速酶AANAT在大鼠老化时的变化。在
前人基础上建立了一种AD大鼠模型并在行为学、组织水平、蛋白水平、
分子水平、超微病理、基因水平和第二信使水平得到了进一步证实。同时
探讨了不同剂量的MT对模型大鼠中枢神经系统AD相关基因APP、PS-1、
PS-2表达和胞内钙的影响，主要结果如下：
一、MT受体和MT限速酶在正常大鼠中表达的时空特点
1、Mel 1a和Mel 1b受体在正常成年大鼠CNS的分布差异（原位杂交）
（1）Mel 1a mRNA-positive细胞主要分布于海马锥体细胞层和齿状回、
额叶和顶叶大脑皮层、视上核、室旁核、视交叉上核、中松果体缰核、小
脑Purkinje细胞层和顶核、脊髓前角、丘脑纹状体髓质、丘脑外侧核、外
侧丘系、内侧丘系、面神经核、巨细胞网状核、三叉神经核和纹状皮质等。
m Mel fo mRNA｛ositive细胞主要分布于小脑 Purkinie细胞层、顶核、
球状核和栓状核，海马锥体细胞层和齿状回、额叶、顶叶、颔叶和枕叶大
脑皮层，脊髓前角，视上核和交叉上核。
m Mel la mRNA中ositive细胞分布广泛、含量丰富，而 Mel fo
mRNA｛ositive细胞分布较为局限，但在海马和大脑皮层这两种受体表达
均非常丰富，提示该区可能是MT的两个主要作用部位。
2、采用RTFCR的方法检测了从胚胎期到老年期不同鼠龄大鼠中枢
AANAT的表达变化。发现胚胎 16天，大脑皮层额叶 AANAT有较高的表
达，生后1周组明显降低，而出生后2周组AANAT表达达到峰值，生后
3周组明显降低，和生后一月、三月组及18月龄老年组无明显差异。在海
马，胚胎 16天，AANAT有较高的表达，生后 1周组降低，生后 2周组表达
达峰值，生后3周组AANAT表达明显降低，和一月龄组、三月龄无明显
差异，18月龄老年组海马 AANAT表达基本消失，提示 AANAT表达的下降
可能是老化时MT水平下降的一个原因。
二、AD大鼠模型的制备及病理学改变
1、AD大鼠的制备，采用皮下间断注射线粒体呼吸链抑制剂叠氮钠，
连续四周，采用MOmS水迷宫对大鼠进行空间记忆能力检测，发现大鼠潜
伏期延长，提示AD大鼠出现记忆功能障碍。
2、电镜观察AD大鼠大脑皮层和海马神经元中超微结构，发现海马和
额叶大脑皮层神经元中出现脂褐素沉积和大量髓鞘样改变，线粒体肿胀，
线粒体峭断裂、减少。突触密度降低，突触内突触小泡数目减少，突触间
隙增宽，突触后致密物厚度降低戾触下致密小体数目明显减少，表明神经
元出现氧化性损伤和老化改变，在超微病理水平证实该AD模型的可靠性。
3、AD大鼠海马、大脑皮层A6在注射NaN3后四周较正常对照组明
显升高，与AD密切相关的基因APP、PSJ、PS上也高于正常对照组，提示
NaN3除了造成神经损伤外，也能导致AD相关基因的活跃表达。
4、AD大鼠模型在注射 NaN3一周组大鼠海马 Mel 3受体表达上调，
在二周和三周组仍过度表达，在四周组达峰值，而 Mel受体表达无明显
Vlll
变化，提示AD大鼠脑内MT受体出现代偿性高表达。
5、在加入 NaN。后 205，海马脑片 CA区锥体细胞明显升高，处于钙
超载状态；加入NaN。后200s，急性分离细胞胞浆内的 KaD］i即开始升高，
并在随后的 10min中持续升高，11 min时上升趋势减缓，提示 NaN。致神经
损伤的最早反应可能是钙超载而升高的钙离子可能来自贮存钙。
三、MT对AD大鼠模型记忆的影响及可能机制
1、MT能显著降低AD大鼠在MOtriS水迷宫测试中的潜伏期，但小剂量
和大剂量MT的作用差异不明显，表明MT可改善AD大鼠的空间记忆能力。
2、不同剂量的MT能阻断AD大鼠大脑皮层和海马中AO的升高，以
IMT、SMT和10MT的作用尤为明显，同时MT也可使AD大鼠大脑皮
层和海马中过度表达的 APP mRNA、PSd 和 PS上 有所下降，
但MT的作用并没有量效关系，提示MT改善AD记忆功能的基础可能是
通过抑制 CNS Ap的生成及 AD相关基因的过度表达。
Alzheimer disease (AD) is a kind of neurodegenerative disease, Which is
characterized with the memory losing in the aged. The morbidity of AD in the
elder is only lower than that of angiocardiopathy and cancer. The pathogeny of
AD is complicated, and the main aspects are included:(l) damage and aging of
neurocyte;(2)inherent causes, some authentic AD related genes including
Amyloid precursor protein(APP), Presenilin- 1 (PS-i), Presenilin-2(PS-2) and
ApoE gene,(3)autoimmunity of nervous system. In the metaphase and
anaphase of AD, the oxidative stress is obvious and outstanding, and the
oxidative stress is an important and key physiopathologic process of AD.
The depress of melatonin (MT) is regarded as an important cause of AD,
which could explain the decrease of noradrenaline and serotonin and the
increase of monoamine oxidase activity in the hippocampus of AD patients.
Much attention was attracted to the foreground of MT in the therapeusis of AD,
for MT is outstanding in these aspects:(1) easy to cross the blood-brain
barrier ;(2) the most powerfiul free radical scavenger by far;(3) the potent
lipotropic antioxidant;(4) inhibition of Alzheimer beta-fibrillogenesis.
With the methods of hybridization in situ, laser scanning confocal
microscope(LSCM), RT-PCR, radioimmunoassay , electron microscope and
Morris water maze test, we observed the distribution of melatonin receptor
Mel la and Mel lb in the CNS of adult rats and the changes of melatonin
II
rate-limiting enzyme arylalkylamine N-acetyltranferase (AANAT) expression
during brain development and aging . And an AD model of rats were
established according to the predecessor and confirmed further in behavior,
tissue, peptide, molecule, ultrastructure, gene and second messenger level. The
effect of melatonin of different dosage on the expression of AANAT and AD
related genes such as APP, PS-i, P5-2 were observed in the CNS of AD
rats.Tbe effect of melatonin on the free introcytoplasmic calcium [Ca2~]i level
of hippocampus slice and isolated pyramidale neurons were also involved in
our experiment. Our results were as follows:
1. Expression of melatonin receptor and its rat-limiting enzyme in the
CNS of normal rats: time and space character
(1) Distribution difference between Mel 1 a and Mel lb receptor mRNA in
the CNS of normal adult rats (hybridization in situ research)
@ Mel la mRNA-positive neurons-rich parts and nucleus of CNS were
as follows: stratum pyramidale and denate gyrus of hippocampus, frontal and
parietal lobe of cortex, supraoptic nucleus, paraventricular nucleus,
suprachiasmatic nucleus, medial habenula2r nucleus, Purkinje cell layer and
fastigial nucleus of cerebellar, anterior horn of the spinal cord, thalamostriate
medulla, ventral lateral nucleus of thalamus, Lemniscus Lateralis, medial
lemniscus, facial nerve nucleus, gigantocellular reticular nucleus, trigeminal
nerve nucleus and striatum cortex etc.
?Mel lb mRNA-positive neurons-rich parts and nucleus of CNS were
as follows: Purkinje cell layer of cerebellar, fastigial nucleus, global nucleus,
emboliform nucleus of the medullaris cerebelli, stratum pyramidale and denate
gyrus of hippocampus, frontal, parietal, occipital and temporal lobe of cortex,
anterior horn of the spinal cord, supraoptic nucleus and suprachiasmatic
nucleus.
?Mel 1 a mRNA-positive neurons were distributed very widely in CNS,
神经变性疾病，在老年人群中的发病率仅次于心脑血管疾病和癌症。AD
的病因是多方面的，最主要有：（1）神经细胞的损伤和老化；（2）遗传易感性，
其中研究得较清楚的几个与AD有关的基因有APP、PS-1、PS-2、ApoE
基因；（3）神经系统自身免疫。AD在发病中后期都出现氧化应激（oxidative
stress），这是AD重要和关键的病理生理过程。
褪黑素（melatonin，MT）的降低被认为是AD发生的一个重要原因，可
以解释AD病人海马去甲肾上腺素、5-HT的降低及单胺氧化酶活性的增加。
MT对AD的治疗作用成为近年的研究热点，这是因为MT具有以下特点：
1、容易通过血脑屏障。2、是目前所知最强效的自由基清除剂。3、是强效
亲脂性抗氧化剂。4、能阻止β-淀粉样蛋白的形成。
本实验采用原位杂交、RT-PCR、激光共聚焦、放免、电镜和Morris
水迷宫行为学检测等方法，观察了MT受体Mel 1a和Mel 1b在正常成年
大鼠中枢神经系统的分布及MT限速酶AANAT在大鼠老化时的变化。在
前人基础上建立了一种AD大鼠模型并在行为学、组织水平、蛋白水平、
分子水平、超微病理、基因水平和第二信使水平得到了进一步证实。同时
探讨了不同剂量的MT对模型大鼠中枢神经系统AD相关基因APP、PS-1、
PS-2表达和胞内钙的影响，主要结果如下：
一、MT受体和MT限速酶在正常大鼠中表达的时空特点
1、Mel 1a和Mel 1b受体在正常成年大鼠CNS的分布差异（原位杂交）
（1）Mel 1a mRNA-positive细胞主要分布于海马锥体细胞层和齿状回、
额叶和顶叶大脑皮层、视上核、室旁核、视交叉上核、中松果体缰核、小
脑Purkinje细胞层和顶核、脊髓前角、丘脑纹状体髓质、丘脑外侧核、外
侧丘系、内侧丘系、面神经核、巨细胞网状核、三叉神经核和纹状皮质等。
m Mel fo mRNA｛ositive细胞主要分布于小脑 Purkinie细胞层、顶核、
球状核和栓状核，海马锥体细胞层和齿状回、额叶、顶叶、颔叶和枕叶大
脑皮层，脊髓前角，视上核和交叉上核。
m Mel la mRNA中ositive细胞分布广泛、含量丰富，而 Mel fo
mRNA｛ositive细胞分布较为局限，但在海马和大脑皮层这两种受体表达
均非常丰富，提示该区可能是MT的两个主要作用部位。
2、采用RTFCR的方法检测了从胚胎期到老年期不同鼠龄大鼠中枢
AANAT的表达变化。发现胚胎 16天，大脑皮层额叶 AANAT有较高的表
达，生后1周组明显降低，而出生后2周组AANAT表达达到峰值，生后
3周组明显降低，和生后一月、三月组及18月龄老年组无明显差异。在海
马，胚胎 16天，AANAT有较高的表达，生后 1周组降低，生后 2周组表达
达峰值，生后3周组AANAT表达明显降低，和一月龄组、三月龄无明显
差异，18月龄老年组海马 AANAT表达基本消失，提示 AANAT表达的下降
可能是老化时MT水平下降的一个原因。
二、AD大鼠模型的制备及病理学改变
1、AD大鼠的制备，采用皮下间断注射线粒体呼吸链抑制剂叠氮钠，
连续四周，采用MOmS水迷宫对大鼠进行空间记忆能力检测，发现大鼠潜
伏期延长，提示AD大鼠出现记忆功能障碍。
2、电镜观察AD大鼠大脑皮层和海马神经元中超微结构，发现海马和
额叶大脑皮层神经元中出现脂褐素沉积和大量髓鞘样改变，线粒体肿胀，
线粒体峭断裂、减少。突触密度降低，突触内突触小泡数目减少，突触间
隙增宽，突触后致密物厚度降低戾触下致密小体数目明显减少，表明神经
元出现氧化性损伤和老化改变，在超微病理水平证实该AD模型的可靠性。
3、AD大鼠海马、大脑皮层A6在注射NaN3后四周较正常对照组明
显升高，与AD密切相关的基因APP、PSJ、PS上也高于正常对照组，提示
NaN3除了造成神经损伤外，也能导致AD相关基因的活跃表达。
4、AD大鼠模型在注射 NaN3一周组大鼠海马 Mel 3受体表达上调，
在二周和三周组仍过度表达，在四周组达峰值，而 Mel受体表达无明显
Vlll
变化，提示AD大鼠脑内MT受体出现代偿性高表达。
5、在加入 NaN。后 205，海马脑片 CA区锥体细胞明显升高，处于钙
超载状态；加入NaN。后200s，急性分离细胞胞浆内的 KaD］i即开始升高，
并在随后的 10min中持续升高，11 min时上升趋势减缓，提示 NaN。致神经
损伤的最早反应可能是钙超载而升高的钙离子可能来自贮存钙。
三、MT对AD大鼠模型记忆的影响及可能机制
1、MT能显著降低AD大鼠在MOtriS水迷宫测试中的潜伏期，但小剂量
和大剂量MT的作用差异不明显，表明MT可改善AD大鼠的空间记忆能力。
2、不同剂量的MT能阻断AD大鼠大脑皮层和海马中AO的升高，以
IMT、SMT和10MT的作用尤为明显，同时MT也可使AD大鼠大脑皮
层和海马中过度表达的 APP mRNA、PSd 和 PS上 有所下降，
但MT的作用并没有量效关系，提示MT改善AD记忆功能的基础可能是
通过抑制 CNS Ap的生成及 AD相关基因的过度表达。
Alzheimer disease (AD) is a kind of neurodegenerative disease, Which is
characterized with the memory losing in the aged. The morbidity of AD in the
elder is only lower than that of angiocardiopathy and cancer. The pathogeny of
AD is complicated, and the main aspects are included:(l) damage and aging of
neurocyte;(2)inherent causes, some authentic AD related genes including
Amyloid precursor protein(APP), Presenilin- 1 (PS-i), Presenilin-2(PS-2) and
ApoE gene,(3)autoimmunity of nervous system. In the metaphase and
anaphase of AD, the oxidative stress is obvious and outstanding, and the
oxidative stress is an important and key physiopathologic process of AD.
The depress of melatonin (MT) is regarded as an important cause of AD,
which could explain the decrease of noradrenaline and serotonin and the
increase of monoamine oxidase activity in the hippocampus of AD patients.
Much attention was attracted to the foreground of MT in the therapeusis of AD,
for MT is outstanding in these aspects:(1) easy to cross the blood-brain
barrier ;(2) the most powerfiul free radical scavenger by far;(3) the potent
lipotropic antioxidant;(4) inhibition of Alzheimer beta-fibrillogenesis.
With the methods of hybridization in situ, laser scanning confocal
microscope(LSCM), RT-PCR, radioimmunoassay , electron microscope and
Morris water maze test, we observed the distribution of melatonin receptor
Mel la and Mel lb in the CNS of adult rats and the changes of melatonin
II
rate-limiting enzyme arylalkylamine N-acetyltranferase (AANAT) expression
during brain development and aging . And an AD model of rats were
established according to the predecessor and confirmed further in behavior,
tissue, peptide, molecule, ultrastructure, gene and second messenger level. The
effect of melatonin of different dosage on the expression of AANAT and AD
related genes such as APP, PS-i, P5-2 were observed in the CNS of AD
rats.Tbe effect of melatonin on the free introcytoplasmic calcium [Ca2~]i level
of hippocampus slice and isolated pyramidale neurons were also involved in
our experiment. Our results were as follows:
1. Expression of melatonin receptor and its rat-limiting enzyme in the
CNS of normal rats: time and space character
(1) Distribution difference between Mel 1 a and Mel lb receptor mRNA in
the CNS of normal adult rats (hybridization in situ research)
@ Mel la mRNA-positive neurons-rich parts and nucleus of CNS were
as follows: stratum pyramidale and denate gyrus of hippocampus, frontal and
parietal lobe of cortex, supraoptic nucleus, paraventricular nucleus,
suprachiasmatic nucleus, medial habenula2r nucleus, Purkinje cell layer and
fastigial nucleus of cerebellar, anterior horn of the spinal cord, thalamostriate
medulla, ventral lateral nucleus of thalamus, Lemniscus Lateralis, medial
lemniscus, facial nerve nucleus, gigantocellular reticular nucleus, trigeminal
nerve nucleus and striatum cortex etc.
?Mel lb mRNA-positive neurons-rich parts and nucleus of CNS were
as follows: Purkinje cell layer of cerebellar, fastigial nucleus, global nucleus,
emboliform nucleus of the medullaris cerebelli, stratum pyramidale and denate
gyrus of hippocampus, frontal, parietal, occipital and temporal lobe of cortex,
anterior horn of the spinal cord, supraoptic nucleus and suprachiasmatic
nucleus.
?Mel 1 a mRNA-positive neurons were distributed very widely in CNS,
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22 Sugden D; McArthur AJ; Ajpru S; et al. Expression of mt(l) melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a
quantitative RT-PCR study across the diurnal cycle.Brain Res Mol Brain Res. 1999; 72(2) : 176-82
23 Fujieda H; Scher J; Hamadanizadeh SA; et al..Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina.Vis Neurosci. 2000 ; 17(1) : 63-70
24 Vanecek J; Watanabe K.Mechanisms of melatonin action in the pituitary and SCN. Adv Exp Med Biol. 1999; 460: 191-8
25 Benot S; Goberna R; Reiter R J;et al.Physiological levels of melatonin contribute to the antioxidant capacity of human serum. J Pineal Res. 1999; 27(1) : 59-64
26 Schmid H A; Requintina P J; Oxenkrug G F;et al.Calcium, calcification, and melatonin biosynthesis in the human pineal gland: a postmortem study into age-related factors.J Pineal Res. 1994; 16(4) : 178-83
27 Humbert W; Pevet P. The decrease of pineal melatonin production with age. Causes and consequences. Ann N Y Acad Sci. 1994; 719: 43-63
28 Benloucif S; Masana M I; Dubocovich M L. Responsiveness to melatonin and its receptor expression in the aging circadian clock of mice. Am J Physiol. 1997 ; 273(6 Pt 2) : R1855-60
29 Quay WB. Circadian rhythem in rat pineal serotonin and its modifications by estrous cycle and photoperiod. Gen. Comp. Endocrinol. 1963: 3:473-79
30 Estrada L; Kanelakis KC; Levy GN;et al..Tissue-and gender-specific expression of N-acetyltransferase 2 (Nat2*) during development of the outbred mouse strain CD-1． Drug Metab Dispos. 2000; 28(2) : 139-46
31 Miguez J M; Recio J; Sanchez Barcelo E; et al.Changes with age in daytime and nighttime contents of melatonin, indoleamines, and catecholamines in the pineal gland: a comparative study in rat and Syrian hamster.J Pineal Res. 1998 ; 25(2) : 106-15