【摘要】：第一部分：GABA能神經(jīng)傳遞紊亂與抑郁癥患者HPA軸過度激活的關(guān)系 目的：下丘腦室旁核(hypothalamic paraventricular nucleus, PVN)中的促腎上腺皮質(zhì)激素釋放激素(corticotropin-releasing hormone, CRH)神經(jīng)元的激活在情感障礙發(fā)病機(jī)制中扮演著至關(guān)重要的作用。γ-氨基丁酸(Gamma-aminobutyric acid, GABA)是主要的抑制性神經(jīng)遞質(zhì)之一。我們的假說是抑郁癥中下丘腦PVN中GABA能神經(jīng)遞質(zhì)失調(diào)可能會(huì)導(dǎo)致CRH免疫反應(yīng)性(immunoreactive, IR)神經(jīng)元的過度激活。方法：通過免疫細(xì)胞化學(xué)和圖像分析方法對(duì)14位情感障礙患者和12位對(duì)照者下丘腦PVN中的谷氨酸脫羧酶(Glutamic acid decarboxylase, GAD65/67)免疫染色進(jìn)行定量分析。14位情感障礙患者中9位患有重性抑郁(major depressive disorder, MDD),5位患有雙向障礙(bipolar disorder, BD)。此外,我們還對(duì)這些個(gè)體的下丘腦PVN中CRH-IR神經(jīng)元總數(shù)進(jìn)行了分析。 結(jié)果：情感障礙患者PVN-GAD65/67呈現(xiàn)神經(jīng)末梢點(diǎn)狀染色分布。GAD65/67-IR結(jié)構(gòu)的密度比對(duì)照組低(P=0.080),在MDD組降低更為顯著(P=0.028),PVN-GAD65/67-IR表達(dá)的下降伴隨著PVN-CRH-IR神經(jīng)元總數(shù)的顯著增加。在情感障礙組中,PVN-GAD65/67-IR結(jié)構(gòu)的密度與CRH-IR神經(jīng)元總數(shù)之間存在顯著負(fù)相關(guān)(rho=-0.527,P=0.032,n=13),該顯著相關(guān)性在對(duì)照組中不存在。 結(jié)論：情感障礙患者(尤其是MDD)PVN中GABA能神經(jīng)支配減弱,降低了GABA對(duì)CRH-IR神經(jīng)元的抑制作用,這是抑郁癥患者HPA軸活性亢進(jìn)的重要機(jī)制之一。 第二部分：NOS-NO系統(tǒng)在應(yīng)激動(dòng)物模型中的初步研究 目的：已有研究表明,氣體性神經(jīng)遞質(zhì)一氧化氮(nitric oxide, NO)及其神經(jīng)元型NO合酶(neuronal NO synthase, nNOS)在抑郁癥患者大腦和血漿中發(fā)生明顯改變,但是它們參與抑郁癥發(fā)病的作用機(jī)制尚不清楚。本文旨在探討NOS-NO系統(tǒng)在應(yīng)激后大鼠下丘腦和血漿中是否改變,從而為將來研究NOS-NO系統(tǒng)參與抑郁癥發(fā)病機(jī)制尋找恰當(dāng)?shù)膭?dòng)物模型和提供科學(xué)依據(jù)。 方法：在成年雄性大鼠建立足底電擊誘導(dǎo)的急性應(yīng)激模型和慢性不可預(yù)知性應(yīng)激(chronic unpredicted stress, CUS)模型。對(duì)照組未受到任何刺激。CUS大鼠先進(jìn)行曠場(chǎng)實(shí)驗(yàn)和糖水偏好實(shí)驗(yàn)后取材。血漿中NO代謝物(硝酸鹽和亞硝酸鹽,NOx)、皮質(zhì)酮(CORT)以及下丘腦NOS活性分別通過各自的商業(yè)化試劑盒進(jìn)行測(cè)定。在下丘腦室旁核(paraventricular nucleus, PVN)中進(jìn)行CRH和nNOS免疫細(xì)胞化學(xué)研究,應(yīng)用圖像分析軟件進(jìn)行定量分析。nNOS與CRH、血管加壓素、催產(chǎn)素等神經(jīng)肽在PVN的共定位關(guān)系通過免疫熒光雙標(biāo)實(shí)驗(yàn)觀察。 結(jié)果：血漿中CORT水平在足底電擊0、5、15和30min后顯著升高(P≤0.038),血漿中NOx水平分別在0和30min出現(xiàn)高峰(P≤0.005)。下丘腦PVN中CRH-IR于足底電擊后15min(P=0.024)和30min(P=0.022)明顯增強(qiáng),而nNOS-IR于足底電擊后15min明顯減弱(P=0.030)。下丘腦總NOS活性在足底電擊后15min明顯下降(P=0.028)。CUS大鼠在曠場(chǎng)和糖水偏好實(shí)驗(yàn)中表現(xiàn)出抑郁焦慮樣行為,血漿中CORT(P=0.001)和NOx(P=0.001)水平均顯著升高,下丘腦總NOS活性在CUS后明顯下降(P=0.025),下丘腦PVN中nNOS陽(yáng)性細(xì)胞密度在CUS組中顯著下降(P=0.019),并且該顯著性降低在nNOS強(qiáng)陽(yáng)性細(xì)胞和nNOS弱陽(yáng)性細(xì)胞上均有所體現(xiàn)。nNOS-IR主要與催產(chǎn)素神經(jīng)元共存,而幾乎不表達(dá)于CRH-IR神經(jīng)元。 結(jié)論：在足底電擊誘導(dǎo)的急性應(yīng)激反應(yīng)和CUS誘導(dǎo)的抑郁癥動(dòng)物模型中,下丘腦總NOS的活性和PVN-nNOS表達(dá)顯著降低,血漿NO水平卻明顯升高。此外,足底電擊后nNOS-IR并不與CRH-IR神經(jīng)元共存,而是主要表達(dá)在催產(chǎn)素細(xì)胞上。因此,nNOS與催產(chǎn)素神經(jīng)元的相互作用將是研究nNOS參與應(yīng)激反應(yīng)機(jī)制的重要線索之一。 第三部分：NOS-NO系統(tǒng)通過改變GABA能神經(jīng)傳遞參與抑郁癥患者前額葉皮層功能調(diào)節(jié) 目的：前額葉皮層(prefrontal cortex, PFC)在結(jié)構(gòu)和功能上的異常與抑郁癥的病因?qū)W和癥狀學(xué)密切相關(guān)。近年來研究表明,氣體性神經(jīng)遞質(zhì)一氧化氮(nitric oxide, NO)在抑郁癥病理過程中發(fā)揮重要作用,但是它是否參與并如何調(diào)節(jié)PFC的活性目前還不清楚。因此,本文旨在研究抑郁癥患者腦脊液(cerebrospinal fluid, CSF)中NO含量和PFC中NO合酶(NO synthase),即神經(jīng)型NOS (neuronal NOS)、內(nèi)皮型NOS (endothelial NOS)和誘導(dǎo)型NOS (iNOS)的表達(dá)。此外,我們還研究了選擇性阻斷nNOS來源的NO產(chǎn)生對(duì)PFC-GABA能神經(jīng)元活性的影響。方法：通過測(cè)定CSF內(nèi)NO代謝產(chǎn)物,即硝酸鹽和亞硝酸鹽(NO、)的含量來反映抑郁癥患者和匹配的對(duì)照者CSF中NO水平。采用實(shí)時(shí)定量PCR技術(shù)檢測(cè)背外側(cè)PFC (dorsolateral PFC, DLPFC)和前扣帶回皮層(anterior cingulate cortex, ACC)中的nNOS.eNOS和iNOS的mRNA水平。應(yīng)用免疫細(xì)胞化學(xué)方法和圖像分析軟件在ACC中定量分析nNOS蛋白水平,并通過免疫熒光雙標(biāo)技術(shù)觀察nNOS與谷氨酸脫羧酶(GAD65/67)的共定位情況。此外,應(yīng)用nNOS的選擇性抑制劑7-nitroindazole(7一NI)處理小鼠ACC腦片,觀察其對(duì)GABA能神經(jīng)元電生理活動(dòng)的改變。 結(jié)果：抑郁癥組CSF-NOx水平顯著低于對(duì)照組(P=0.007)。抑郁癥患者ACC中,而不是DLPFC中,nNOS-mRNA水平具有降低的趨勢(shì)(P=0.083).nNOS-IR王要分布在正常人腦ACC的Ⅱ/Ⅲ層。在抑郁癥患者組中,ACC-nNOS-IR在整個(gè)灰質(zhì)中的細(xì)胞密度和平均光密度均降低(P=0.083),尤其是ACC的Ⅱ/Ⅲ層降低更為顯著(P=0.043)。對(duì)照組nNOS-IR在ACC灰質(zhì)中的細(xì)胞密度與CSF-NOx水平呈顯著正相關(guān)(rho=0.667,P=0.050,n=9).nNOS與GABA能神經(jīng)元的標(biāo)記物GAD65/67在抑郁癥患者ACC中存在明顯共定位。此外,nNOS選擇性抑制劑7-NI與ACC腦片孵育顯著增加了GABA介導(dǎo)的抑制性突觸后電流(mIPSCs)的頻率(P0.05, n=11),但未改變mIPSCs的幅度。 結(jié)論：抑郁癥患者ACC-nNOS表達(dá)下調(diào),CSF-NOx水平減低,選擇性抑制小鼠ACC中的nNOS活性顯著影響了GABA能神經(jīng)傳遞。
[Abstract]:Part one: the relationship between GABA nerve transmission disturbance and HPA axis overactivation in depressive patients
AIM: Activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) plays an important role in the pathogenesis of affective disorders. Gamma-aminobutyric acid (GABA) is the main inhibitory agent. Our hypothesis is that the imbalance of GABAergic neurotransmitters in hypothalamic PVN in depression may lead to excessive activation of CRH immunoreactive (IR) neurons. METHODS: Glutamate decarboxylase (G-decarboxylase) in hypothalamic PVN was detected by immunocytochemistry and image analysis in 14 patients with affective disorders and 12 controls. Immunostaining with lutamic acid decarboxylase (GAD65/67) was used to quantitatively analyze the number of CRH-IR neurons in PVN of hypothalamus in 9 of 14 patients with affective disorder (MDD) and 5 with bipolar disorder (BD).
Results: The distribution of PVN-GAD65/67 in patients with affective disorders was dot-like. The density of PVN-GAD65/67-IR structure was lower than that of the control group (P=0.080), and the decrease of PVN-GAD65/67-IR expression was more significant in the MDD group (P=0.028). The decrease of PVN-GAD65/67-IR expression was accompanied by a significant increase of the total number of PVN-CRH-IR neurons. There was a significant negative correlation between the total number of CRH-IR neurons (rho=-0.527, P=0.032, n=13), which did not exist in the control group.
Conclusion: The decrease of GABAergic innervation in PVN of patients with affective disorders (especially MDD) can decrease the inhibitory effect of GABA on CRH-IR neurons, which is one of the important mechanisms of HPA axis hyperactivity in depressive patients.
The second part: a preliminary study of NOS-NO system in stress animal models.
AIM: Previous studies have shown that nitric oxide (NO) and its neuronal nitric oxide synthase (nNOS) are significantly altered in the brain and plasma of depressive patients, but the mechanism of their involvement in the pathogenesis of depression is still unclear. Whether the hypothalamus and plasma are altered provides a scientific basis for the future study of NOS-NO system involved in the pathogenesis of depression.
METHODS: Acute and chronic unpredictable stress (CUS) models induced by plantar electric shock were established in adult male rats. The control group was not stimulated by any stimulation. After open-field experiment and sugar preference experiment, the plasma NO metabolites (nitrate and nitrite, NO), corticosterone (C The activity of NOS in hypothalamus and ORT was measured by commercial kits. CRH and nNOS immunocytochemistry were studied in paraventricular nucleus (PVN) of hypothalamus, and quantitative analysis was performed by image analysis software. Experimental observation of double fluorescence of disease.
Results: The plasma CORT level increased significantly (P < 0.038) at 0, 5, 15 and 30 minutes after plantar shock, and the plasma NOX level peaked at 0 and 30 minutes respectively (P < 0.005). CRH-IR in hypothalamic PVN increased significantly at 15 minutes (P = 0.024) and 30 minutes (P = 0.022) after plantar shock, while nNOS-IR decreased significantly at 15 minutes (P = 0.030). The activity of total NOS in hypothalamus decreased significantly after CUS (P = 0.025) and the density of nNOS positive cells in PVN in hypothalamus decreased significantly (P = 0.025) in CUS group. NNOS-IR mainly coexisted with oxytocin neurons, but hardly expressed in CRH-IR neurons.
CONCLUSION: Total NOS activity and PVN-nNOS expression in hypothalamus were significantly decreased and plasma NO level was significantly elevated in rats with acute stress induced by plantar electric shock and depression induced by CUS. In addition, nNOS-IR did not coexist with CRH-IR neurons, but mainly expressed in oxytocin cells after plantar electric shock. Neuronal interaction is one of the important clues to study the mechanism of nNOS involved in stress response.
Part III: NOS-NO system participates in the regulation of prefrontal cortex function in depressive patients by altering GABAergic neurotransmission
Objective: The structural and functional abnormalities of prefrontal cortex (PFC) are closely related to the etiology and symptoms of depression. Recent studies have shown that nitric oxide (NO) plays an important role in the pathological process of depression, but whether it participates in and how to regulate the activity of PFC. Therefore, the aim of this study was to investigate the content of NO in cerebrospinal fluid (CSF) and the expression of NO synthase (NOS), endothelial NOS and inducible NOS (iNOS) in PFC-GABA in depressed patients. Methods: The levels of NO in CSF of depressive patients and matched controls were measured by measuring the contents of nitrate and nitrite metabolites in CSF. Real-time quantitative PCR was used to detect dorsolateral PFC (DLPFC) and anterior cingulate cortex (ACC). The levels of nNOS, eNOS and iNOS mRNA were measured by immunocytochemistry and image analysis software in ACC. The co-localization of nNOS and glutamate decarboxylase (GAD65/67) was observed by immunofluorescence double labeling technique. In addition, the selective inhibitor of nNOS, 7-nitroindazole (7-NI), was used to treat ACC slices of mice. The changes in electrophysiological activity of GABA neurons were observed.
Results: The level of CSF-NOx in depression group was significantly lower than that in control group (P = 0.007). In ACC, but not DLPFC, the level of nNOS-mRNA was decreased (P = 0.083). The nNOS-IR kingdom was distributed in layer II/III of ACC in normal human brain. In depression group, the cell density and average optical density of ACC-nNOS-IR in the whole gray matter were decreased (P = 0.083). In control group, the cell density of nNOS-IR in ACC gray matter was positively correlated with CSF-NOx level (rho = 0.667, P = 0.050, n = 9). There was significant co-localization between nNOS and GABAergic neuron marker GAD65/67 in ACC of depressive patients. Slice incubation significantly increased the frequency of GABA-mediated inhibitory postsynaptic currents (mIPSCs) (P 0.05, n=11), but did not change the amplitude of mIPSCs.
CONCLUSION: The expression of ACC-nNOS is down-regulated and the level of CSF-NOx is down-regulated in depressed patients. Selective inhibition of nNOS activity in ACC of mice significantly affects GABAergic neurotransmission.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.4

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 趙玲蘢;補(bǔ)心寧神法治療心膽氣虛型驚恐障礙近期臨床療效觀察[D];北京中醫(yī)藥大學(xué);2014年

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本文編號(hào)：2218840