【作者】 张金玲；

【导师】 许崇涛；

【作者基本信息】 汕头大学 ， 精神病与精神卫生学， 2007， 硕士

【摘要】 背景：腺苷是一种睡眠因子，它主要通过其受体来调节睡眠。其中，腺苷A₁受体起主要作用。此外，腺苷作为一种神经调质，可以通过其受体尤其是腺苷A₁受体调节各种神经递质的释放。最近又有研究发现腺苷及其受体与抑郁及抗抑郁过程有一定联系。而睡眠剥夺可发挥快速抗抑郁效应，但其中的神经生物机制至今未明，目前的研究多集中于单胺类神经递质系统。既然腺苷与睡眠、抑郁症及各种神经递质均有密切关系，那么腺苷是否参与了睡眠剥夺的快速抗抑郁过程呢?在此过程中，腺苷受体的表达是否有所变化呢?目前尚无这方面的报道。本课题在研究正常大鼠连续72小时快眼动睡眠剥夺（rapid eye movement sleep deprivation,REMSD）后腺苷A₁受体和5-HT_1A受体表达及神经元活性改变的基础上，并与抑郁模型大鼠连续72小时快眼动睡眠剥夺后大鼠不同脑区腺苷A₁受体和5-HT_1A受体表达变化进行比较，探讨腺苷A₁受体在睡眠剥夺快速抗抑郁机制中可能的作用。实验一：目的：观察睡眠剥夺对正常大鼠自发活动和不同脑区腺苷A₁受体和5-HT_1A受体表达的影响及对神经元活性的影响。材料和方法：实验动物为Sprague Dawley 2-3月龄雄性大鼠，随机分为正常+72小时REMSD组（n=8，以下称睡眠剥夺组）、正常+大平台对照组（n=8，以下称大平台对照组）、正常对照组（n=8，以下称正常对照组）。快眼动睡眠剥夺采用小平台水环境法；用大平台水环境做对照，排除水环境的影响。观察快眼动睡眠剥夺作用下大鼠自发活动变化，采用逆转录-聚合酶链反应法（reverse transcription-polymerase chain reaction,RT-PCR）检测各组大鼠海马、前额皮质、中脑、下丘脑、纹状体腺苷A₁受体和5-HT_1A受体mRNA表达的变化，运用尼氏染色技术观察神经元的活性变化。实验结果用Excel 2000和SPSS 11.0统计软件进行分析处理。结果以均数±标准误（X±SE）表示，采用单因素方差分析（ANOVA）和t检验（T-test）进行组间均数比较，P＜0.05为差异显著，P＜0.01为差异有高度显著性。结果：1．正常大鼠经72小时REMSD后，自发活动总路程较大平台对照组增加（P＜0.05）；自发活动中央路程与休息时间均较大平台对照组无明显变化（P＞0.05）。正常大鼠经大平台水环境处理后，自发活动总路程、中央路程及休息时间较正常对照组均无明显变化（P＞0.05）。2．正常大鼠经72小时REMSD后与大平台组比较海马腺苷A₁受体mRNA转录水平显著降低（P＜0.01），而5-HT_1A受体mRNA转录水平升高（P＜0.05）；大平台对照组经72小时水环境处理使正常大鼠海马腺苷A₁受体mRNA转录水平显著升高（P＜0.01），而5-HT_1A受体mRNA转录水平无明显变化（P＞0.05）。3．72小时REMSD本身不能使正常大鼠前额皮质腺苷A₁受体mRNA表达发生明显变化（P＞0.05），而可使5-HT_1A受体mRNA表达升高（P＜0.05）；72小时大平台水环境处理使正常大鼠前额皮质腺苷A₁受体mRNA表达降低（P＜0.05）；而对前额皮质5-HT_1A受体mRNA表达无明显影响（P＞0.05）。4．正常大鼠经72小时REMSD后，与大平台对照组比中脑腺苷A₁受体mRNA表达与5-HT_1A受体mRNA表达均降低（P＜0.05）；72小时大平台水环境处理后，正常大鼠中脑腺苷A₁受体mRNA表达降低（P＜0.05），而中脑5-HT_1A受体mRNA表达升高（P＜0.05）。5．72小时REMSD本身对正常大鼠下丘脑腺苷A₁受体及5-HT_1A受体mRNA表达均无明显影响（P＞0.05）；72小时大平台水环境处理也未改变正常大鼠下丘脑腺苷A₁受体mRNA表达（P＞0.05），却可使下丘脑5-HT_1A受体mRNA表达升高（P＜0.05）。6．72小时REMSD及72小时大平台水环境处理均未改变纹状体腺苷A₁受体及5-HT_1A受体mRNA表达（P＞0.05）。7．尼氏染色结果显示，正常大鼠经72小时睡眠剥夺后与大平台对照组比海马尼氏小体染色光密度升高（P＜0.05）；大平台对照组与正常对照组比海马尼氏小体染色光密度无明显变化（P＞0.05）。实验二：目的：观察睡眠剥夺对慢性应激抑郁模型大鼠自发活动和不同脑区腺苷A₁受体和5-HT_1A受体表达的影响。材料和方法：实验动物为Sprague Dawley 2-3月龄雄性大鼠，随机分为正常对照组（n=10，以下称正常对照组）、应激造模组（n=48）。应激造模组采用慢性轻度不可预见性应激（chronic mild unpredicted stress,CMUS）和分养两种经典模型结合的方式来建立抑郁模型，采用随机区组法把造模成功大鼠分为3组：抑郁模型组（n=8，以下称抑郁模型组）、抑郁模型+72小时REMSD组（n=8，以下称睡眠剥夺组）、抑郁模型+大平台对照组（n=7，以下称大平台对照组）。利用小平台水环境法对抑郁模型大鼠进行快眼动睡眠剥夺，并用大平台水环境做对照。观察快眼动睡眠剥夺作用下抑郁模型大鼠自发活动变化，采用逆转录-聚合酶链反应法（reverse transcription-polymerase chain reaction,RT-PCR）检测各组大鼠前额皮质、海马、下丘脑、中脑各脑区腺苷A₁受体和5-HT_1A受体mRNA含量的变化，运用免疫组化技术对腺苷A₁受体蛋白表达进行定位和分析。实验结果用Excel 2000和SPSS 11.0统计软件进行分析处理。结果以均数±标准误（X±SE）表示，采用单因素方差分析（ANOVA）和t检验（T-test）进行组间均数比较，P＜0.05为差异显著。结果：1．大鼠经过21天慢性轻度不可预见性应激处理后，自发活动总路程、中央路程较正常对照组显著减少，休息时间延长（P＜0.05）；抑郁模型大鼠经过72小时REMSD后，自发活动总路程、中央路程与大平台对照组相比显著增加，休息时间缩短（P＜0.05）。（该部分是由本专业2003级研究生张印南进行的实验）2．21天慢性应激后，大鼠海马腺苷A₁受体mRNA转录水平与正常对照组相比明显升高（P＜0.01）；72小时REMSD后海马腺苷A₁受体mRNA表达与大平台对照组相比降低（P＜0.05）；72小时大平台水环境处理对抑郁模型大鼠海马腺苷A₁受体mRNA转录水平没有影响（P＞0.05）。3．21天慢性应激可使正常大鼠前额皮质腺苷A₁受体mRNA转录水平显著下降（P＜0.01）；而72小时REMSD对这种变化影响不明显（P＞0.05）；72小时大平台水环境处理对这种变化亦无明显影响（P＞0.05）。4．21天慢性应激不能明显改变正常大鼠中脑部位腺苷A₁受体mRNA转录水平（P＞0.05）；72小时REMSD及水环境本身对抑郁模型大鼠中脑部位腺苷A₁受体mRNA转录水平均无明显影响（P＞0.05）。5．21天慢性应激之后，正常大鼠下丘脑部位腺苷A₁受体mRNA转录水平与正常对照组相比差别无统计学意义（P＞0.05）；72小时REMSD后腺苷A₁受体mRNA转录水平与大平台组相比有所增加（P＜0.05），72小时大平台水环境处理对抑郁模型大鼠下丘脑腺苷A₁受体mRNA表达无影响（P＞0.05）。6．免疫组化结果显示腺苷A₁受体阳性细胞主要分布在海马，小脑等脑区，其他脑区分布很少。21天慢性应激后，大鼠海马腺苷A₁受体蛋白表达水平与正常对照组相比升高（P＜0.05）；72小时REMSD后海马腺苷A₁受体表达与大平台组相比降低（P＜0.05）；大平台对照组经72小时水环境处理对抑郁模型大鼠海马腺苷A₁受体蛋白表达水平没有影响（P＞0.05）。7．睡眠剥夺对慢性应激抑郁模型大鼠不同脑区5-HT_1A受体表达的影响结果见附录（该部分试验和分析是由张印南师兄进行的）结论：72小时REMSD可快速逆转21天慢性应激所导致行为性抑郁。海马腺苷A₁受体mRNA转录水平和蛋白表达水平升高和前额皮质腺苷A₁受体mRNA转录水平降低可能参与了抑郁症的发病过程。而在睡眠剥夺抗抑郁的机制中，可能海马腺苷A₁受体mRNA转录水平和蛋白表达水平恢复性降低和下丘脑腺苷A₁受体mRNA转录水平的升高发挥重要作用。腺苷A₁受体在睡眠剥夺快速抗抑郁过程中的作用可能与5-HT系统有关。更多还原

【Abstract】 Background: Adenosine is thought as a sleep factor, which modulates sleep viaits receptors. Adenosine A₁ receptor plays a key role in sleep process. Furthermore,adenosine also modulates some neurotransmitters release as a neuromodulator.Recently a few studies indicated that adenosine and its receptors related withdepression and antidepressant. It is known that sleep deprivation presents anantidepressant-like effect. However, the mechanism of the rapid antidepressanteffect of sleep deprivation has not been clarified. Although most of the studiesof sleep deprivation focus on monoamine system, there still is evidence indicatedthat adenosine system involved in sleep deprivation. Review all of the progressin this field, We hypotheses that adenosine system plays an important role inantidepressant mechanism of sleep deprivation. The questions remained are, Howadenosine levels change in different part of brain when receiving sleep deprivation,The relationship of adenosine levels with antidepressant effect, and which kindof adenosine receptors involved in the process. This study try to investigate themechanism of the antidepressant effects of sleep deprivation via adenosine passway in rats.Study One:Objective: To investigate the effect of SD on locamotor activities and expressionof adenosine A₁ receptor, 5-HT_1A receptor in corresponding brain. To analyze therelationship between adenosine A, receptor and antidepressant effect of SD.Materials and Methods: Male Sprague-Dawley rats were divided into threegroups randomly: 1) normal control rats, 2) sleep deprivation, 3) sleep deprivationcontrol. Locamotor activities were performed in the 0,3rd day of the experiment.Animals were sacrificed at the third day of the experiment, the expression levelof adenosine A₁ receptor and 5-HT_1A receptor mRNA were studied by reversetranscription polymerase chain reaction （RT-PCR）. The vitality of hippocampusnerve was tested by Nissle’s staining. Results: 1) Following 72-hour sleep deprivation, locamotor activities increasedin SD group（P＜0.05, compared with SD control）. 2) Sleep deprivation decreased thelevel of adenosine A₁ receptor in the hippocampus and midbrain （P＜0.05）. sleepdeprivation had no effect on the the level of adenosine Am receptor mRNA inprefrontal cortex, hypothalamus and striate（P＞0.05）. 3) Sleep deprivationincreased the level of 5-HT_1A receptor in the hippocampus and in prefrontalcortex（P＜0.05）. Sleep deprivation decreased the level of 5-HT_1A receptor inmidbrain（P＜0.05）. Seep deprivation had no effect on the the level of 5-HT_1A receptormRNA in hypothalamus and striate（P＞0.05）. 4) Nissle’s staining indicate that Sleepdeprivation increased the vitality of nerves in hippocampus（P＜0.05）.Study Two:Objective: To investigate the effects of an 72-hour REM （rapid eye movement） sleepdeprivation on chronic mild unpredictable stress （CMUS） animal model and theexpression of adenosine A₁ receptor and 5-HT_1A receptor in the brain in chronic mildunpredictable stress （CMUS） animal model and after sleep deprivation.Materials and Methods: Sprague-Dawley rats were divided into two groupsrandomly: 1) normal control rats, 2) CMUS depressant animal model. Then after 21dCMUS, the depressant animal model were divided into three groups randomly: 3) thedepressant animal model,4) 72-hours REM sleep deprivation, 5) tank controlrats, Spontaneous movements were performed in the 0, 21th, 24th day of theexperiment. Animals were sacrificed, the expression level of adenosine A, receptorand 5-HT_1A receptor mRNA were studied by reverse transcription polymerase chainreaction （RT-PCR）. the expression level of adenosine A₁ recetor protein werestudied by immunohistochemistry.Results: 1) Locamotor activities significantly slowed down in the CMUS depressantanimal model, compared with normal control rats（P＜0.05）. Following 72-hour sleepdeprivation, locamotor activities increased in REMSD group（P＜0.05）. （done by ZhangYinnan） 2) Chronic mild unpredictable stress can increase the level of adenosineA₁ receptor（mRNA and protein） in the hippocampus（P＜0.05）, Sleep deprivation decreased the level of adenosine A₁ receptor（mRNA and protein） in thehippocampus（P＜0.05）. 3) Chronic mild unpredictable stress can decrease the levelof adenosine A, receptor mRNA in prefrontal cortex（P＜0.05）, but Sleep deprivationcannot come back the level of adenosine A₁ receptor mRNA in prefrontalcortex（P＞0.05）. 4) Chronic mild unpredictable stress had no effect on the levelof adenosine A, receptor mRNA in hypothalamus（P＞0.05）, but Sleep deprivation canincrease the level of adenosine A₁ receptor mRNA in hypothalamus（P＜0.05）. 5)Chronic mild unpredictable stress and sleep deprivation had no effect on the thelevel of adenosine A₁ receptor mRNA in midbrain（P＞0.05）. 6) The changes of 5-HT_1Areceptor mRNA expression are in the appendix（Done and analysed by Zhang Yinnan）.Conclusions: sleep deprivation has an rapid antidepressant effect for CMUSdepressant rats. The up regulation of the expression of adenosine A₁ receptor inhippocampus and the down regulation of the expression of adenosine A₁ receptor inprefrontal cortex may take part in the pathogenesis of depression. Down regulationof the expression of adenosine A₁ receptor in hippocampus by REM sleep deprivationmay participate in the antidepressant-like effect of sleep deprivation. The upregulation of the expression of adenosine A₁ receptor in hypothalamus may also havean important effect. There may be some relation between adenosine A₁ receptor and5-HT in this course.更多还原