發(fā)布時(shí)間：2018-07-22 11:10

【摘要】： 背景與目的 組胺(Histamine, HA)是人體內(nèi)重要的自體活性物質(zhì),具有廣泛的生理作用,同時(shí)也參與介導(dǎo)多種疾病的病理生理過(guò)程。在中樞神經(jīng)系統(tǒng),存在完整的HA能神經(jīng)體系,HA作為中樞神經(jīng)遞質(zhì)參與介導(dǎo)認(rèn)知記憶、睡眠覺(jué)醒、飲水?dāng)z食等功能。在外周組織中,HA主要以肥大細(xì)胞源和非肥大細(xì)胞源兩種形式貯存。肥大細(xì)胞來(lái)源的HA參與介導(dǎo)變態(tài)反應(yīng)的病理過(guò)程,非肥大細(xì)胞源性HA的生理和病理作用尚不十分清楚。業(yè)已發(fā)現(xiàn),非肥大細(xì)胞來(lái)源的HA存在于大鼠外周胃腸道的神經(jīng)、頸上神經(jīng)節(jié)(Superior cervical ganglion, SCG)、頸動(dòng)脈體感覺(jué)神經(jīng)元等,提示HA可能與這些神經(jīng)元的活動(dòng)有關(guān)。我們以往的研究發(fā)現(xiàn),在豚鼠SCG的神經(jīng)元內(nèi),參與合成HA的組胺酸脫羧酶(Histidine decarboxylase, HDC)與合成去甲腎上腺素(Norepinephrine, NE)的酪氨酸羥化酶(Tyrosine hydroxylase, TH)和多巴胺β羥化酶(Dobamine-β-hydroxylase, DβH)共存,而且HA和NE也共存其中;刺激交感神經(jīng)末梢能使HA和NE從交感神經(jīng)末梢共釋放;激動(dòng)交感神經(jīng)末梢突觸前膜HA H3受體能夠負(fù)反饋調(diào)控HA和NE的釋放。根據(jù)以上研究結(jié)果,我們首先提出HA很可能是一種尚未被認(rèn)識(shí)的新的交感神經(jīng)遞質(zhì),并提出HA參與負(fù)反饋調(diào)控交感神經(jīng)傳遞的假說(shuō)。然而HA與NE在交感神經(jīng)元內(nèi)的共存是否具有普遍性?HA在交感神經(jīng)元內(nèi)的亞細(xì)胞定位如何?HA從交感神經(jīng)元內(nèi)釋放是否遵循經(jīng)典神經(jīng)遞質(zhì)囊泡釋放的規(guī)律?這些都是為最終定義HA作為一種新的交感神經(jīng)遞質(zhì)所必須回答的問(wèn)題�；谝陨蠁�(wèn)題,本研究采用免疫熒光多重標(biāo)記、順行示蹤及激光共聚焦顯微鏡技術(shù),觀察HA在不同種屬動(dòng)物交感神經(jīng)節(jié)與交感神經(jīng)末梢的分布,及其與NE的共存;采用包埋前免疫電鏡技術(shù)觀察HA在交感神經(jīng)末梢和培養(yǎng)交感神經(jīng)節(jié)細(xì)胞的亞細(xì)胞定位;在新生豚鼠原代培養(yǎng)SCG細(xì)胞上,利用FM1-43熒光染料研究HA突觸囊泡釋放、運(yùn)動(dòng)的循環(huán)動(dòng)力學(xué),為最終證實(shí)HA是一種新的交感神經(jīng)遞質(zhì)提供新證據(jù)。開(kāi)展這一領(lǐng)域的研究對(duì)于揭示交感神經(jīng)的新功能,深入認(rèn)識(shí)交感神經(jīng)功能紊亂疾病相關(guān)疾病的發(fā)病機(jī)理,尋找新的具有潛在治療可能性的藥物新靶點(diǎn)等都具有重要的理論意義。 結(jié)果 1.組胺和NE在不同動(dòng)物交感神經(jīng)系統(tǒng)的共存分布 本研究采用免疫熒光雙標(biāo)和激光共聚焦顯微鏡技術(shù),發(fā)現(xiàn)在小鼠SCG、狗SCG和腹腔交感神經(jīng)節(jié)神經(jīng)元內(nèi),以及小鼠和豚鼠輸精管、腸系膜動(dòng)脈的交感神經(jīng)神經(jīng)末梢中均有HA和NE共存。在小鼠和狗的SCG中,NE樣免疫陽(yáng)性神經(jīng)元中HA的雙標(biāo)率分別為70%和40%,狗腹腔節(jié)中NE樣免疫陽(yáng)性神經(jīng)元中HA的雙標(biāo)率為60%。在小鼠和豚鼠的輸精管、腸系膜動(dòng)脈標(biāo)本上,NE樣免疫陽(yáng)性神經(jīng)纖維中HA的雙標(biāo)率分別為80%和30%。在豚鼠SCG局部微量注射順行示蹤劑生物素化葡聚糖胺(Biotinylated dextranamine, BDA),取豚鼠心臟標(biāo)本,采用免疫熒光三重標(biāo)記技術(shù)觀察自SCG投射到心臟的交感神經(jīng)纖維和末梢中HA。結(jié)果顯示,BDA順行示蹤投射到心臟的交感神經(jīng)纖維中,20%的示蹤神經(jīng)纖維呈HA樣免疫陽(yáng)性反應(yīng),10%示蹤神經(jīng)纖維呈現(xiàn)HA和NE樣免疫陽(yáng)性反應(yīng)。上述結(jié)果在細(xì)胞水平確定HA廣泛分布于不同種屬動(dòng)物的交感神經(jīng)系統(tǒng)內(nèi),并與NE共存,為定義HA作為新的交感神經(jīng)遞質(zhì)提供了直接的形態(tài)學(xué)證據(jù)。 2.組胺在交感神經(jīng)末梢及培養(yǎng)SCG神經(jīng)元中的亞細(xì)胞定位 在支配豚鼠輸精管交感神經(jīng)末梢內(nèi),以及原代培養(yǎng)的豚鼠SCG神經(jīng)元內(nèi),利用HA包埋前免疫電鏡技術(shù),電鏡觀察顯示,HA樣免疫陽(yáng)性物質(zhì)定位于直徑約50 nm的小囊泡內(nèi),且HA陽(yáng)性囊泡約占囊泡總數(shù)的90%以上,大囊泡(直徑100 nm)及個(gè)別小囊泡無(wú)HA免疫陽(yáng)性物質(zhì)。結(jié)果表明,交感神經(jīng)元內(nèi)HA主要存在于小囊泡中,本項(xiàng)結(jié)果在亞細(xì)胞水平為定義HA作為新的交感神經(jīng)遞質(zhì)提供了直接的形態(tài)學(xué)證據(jù)。 3.培養(yǎng)SCG神經(jīng)突起部位組胺突觸囊泡循環(huán)動(dòng)力學(xué) 利用FM1-43熒光染料,結(jié)合HA免疫熒光染色,在培養(yǎng)豚鼠SCG神經(jīng)元突起上觀察HA囊泡的釋放動(dòng)力學(xué)和HA囊泡的運(yùn)動(dòng)過(guò)程,激光共聚焦顯微鏡成像并統(tǒng)計(jì)分析結(jié)果。脫色(Destaining)研究結(jié)果顯示,SCG神經(jīng)突起中HA的紅色熒光信號(hào)強(qiáng)度與FM1-43綠色熒光信號(hào)強(qiáng)度隨除極刺激作用時(shí)間的增加,而呈現(xiàn)同時(shí)逐漸減弱的現(xiàn)象,兩者呈顯著正相關(guān)(r = 0.989, P 0.01),表明HA的釋放遵循囊泡釋放動(dòng)力學(xué)的普遍規(guī)律。在除極作用的不同時(shí)間點(diǎn)固定細(xì)胞,分別測(cè)定代表HA的紅色熒光強(qiáng)度和代表FM1-43的綠色熒光強(qiáng)度,結(jié)果表明HA的紅色熒光強(qiáng)度要強(qiáng)于FM1-43的綠色熒光強(qiáng)度。由于在本研究采用的除極條件下,FM1-43熒光信號(hào)主要反映循環(huán)囊泡的釋放情況,HA與FM1-43的熒光強(qiáng)度出現(xiàn)差異的結(jié)果提示,在神經(jīng)突起部位貯存HA的囊泡可能存在可釋放囊泡庫(kù)和儲(chǔ)備囊泡庫(kù)之分。熒光漂白恢復(fù)(Fluorescence recovery after photobleaching, FRAP)結(jié)合免疫組織化學(xué)研究結(jié)果顯示,在SCG神經(jīng)突起處含有HA囊泡的擴(kuò)散速度為0.09μm2/s,其他囊泡為0.08μm2/s。HA囊泡的運(yùn)動(dòng)過(guò)程與其他囊泡無(wú)顯著性差異。 結(jié)論: 1. HA普遍存在于多種動(dòng)物的交感神經(jīng)系統(tǒng),并與經(jīng)典交感神經(jīng)遞質(zhì)NE共存。 2. HA主要貯存于交感神經(jīng)突觸小囊泡內(nèi)。 3.交感神經(jīng)元內(nèi)HA囊泡的釋放遵循經(jīng)典遞質(zhì)囊泡循環(huán)動(dòng)力學(xué)規(guī)律,貯存HA的囊泡可能存在可釋放囊泡庫(kù)和儲(chǔ)備囊泡庫(kù)之分。
[Abstract]:Background and purpose
Histamine (HA) is an important autoactive substance in the human body. It has extensive physiological function and also participates in the pathophysiological process of mediating various diseases. In the central nervous system, there is a complete HA energy system, and HA as a central neurotransmitter is involved in mediating cognitive memory, sleeping awakening, drinking water intake and so on. In the peripheral group In the fabric, HA is mainly stored in two forms of mast cell source and non mast cell source. Mast cell derived HA is involved in the pathological process of mediating allergy. The physiological and pathological effects of non mast cell derived HA are not very clear. It has been found that non mast cell derived HA exists in the peripheral nerve of the stomach and intestines of the rat, and the superior cervical ganglion (Su Perior cervical ganglion, SCG), carotid body sensory neurons, etc., suggesting that HA may be associated with the activity of these neurons. Our previous study found that in the neurons of SCG of the guinea pig, the histamine acid decarboxylase (Histidine decarboxylase, HDC) and the tyrosine hydroxylase synthase of noradrenaline (Norepinephrine, NE) were involved in the SCG of the guinea pig. (Tyrosine hydroxylase, TH) coexist with the dopamine beta hydroxylase (Dobamine- beta -hydroxylase, D beta H), and both HA and NE coexist. Stimulating sympathetic nerve endings can release HA and NE from the sympathetic nerve endings. We first suggested that HA is likely to be a new sympathic neurotransmitter that has not yet been recognized, and proposes the hypothesis that HA participates in the negative feedback regulation of sympathetic neurotransmission. However, does the coexistence of HA and NE in sympathetic neurons are universal? What is the subcellular localization of HA in sympathetic neurons and whether HA is released from sympathetic neurons to follow the classics The release of neurotransmitter vesicles? These are questions that must be answered by the final definition of HA as a new sympathetic neurotransmitter. Based on the above problems, this study uses multiple immunofluorescence markers, anterograde tracing and laser confocal microscopy, to observe the sympathetic ganglion and sympathetic nerve endings of HA in different species of animals. Distribution and coexistence with NE; the subcellular localization of HA in sympathetic nerve endings and cultured sympathetic ganglion cells was observed by pre embedding immunoelectron microscopy, and on the primary cultured SCG cells of newborn guinea pigs, FM1-43 fluorescent dyes were used to study the release of HA synaptic vesicles and the kinetics of movement, which finally confirmed that HA was a new sympathetic nerve. The research in this field is of great theoretical significance to reveal the new function of sympathetic nerve, to understand the pathogenesis of the disease related to the disorder of sympathetic nervous disorder, and to find new potential therapeutic targets for potential treatment.
Result
1. coexistence of histamine and NE in the sympathetic nervous system of different animals
In this study, immunofluorescence double labeling and laser confocal microscopy were used to detect the coexistence of HA and NE in the sympathetic nerve endings of mice and guinea pigs and the sympathetic nerve terminals of the mesenteric arteries in mouse SCG, dog SCG and abdominal sympathetic ganglion neurons, and in the SCG of mice and dogs, the double standard rate of HA in the NE like immunoreactive neurons was respectively in mice and dogs. For 70% and 40%, the double standard rate of HA in NE like immun-positive neurons in the abdominal segment of the dog was 60%. in the vas deferens of mice and guinea pigs, and on the mesenteric artery specimens, the double standard rate of HA in the NE like immun-positive nerve fibers was 80% and 30%. respectively in the local microinjection of SCG in guinea pig SCG (Biotinylated dextranamine, BDA). A guinea pig heart specimen was taken to observe the HA. results in the sympathetic fibers and terminals projected from SCG to the heart by immunofluorescence three markers. The results showed that the BDA tracer projected into the sympathetic fibers of the heart, 20% of the tracer fibers were HA like immunoreactive, and 10% of the tracer fibers presented HA and NE like immunoreaction. The results indicated that HA was widely distributed in the sympathetic nervous system of different species of animals at the cellular level, and coexisted with NE, providing a direct morphological evidence for the definition of HA as a new sympathetic neurotransmitter.
2. subcellular localization of histamine in sympathetic nerve terminals and cultured SCG neurons
In the innervation of the sympathetic nerve endings of the guinea pig's vas deferens and the primary cultured guinea pig SCG neurons, the immuno electron microscopy showed that the HA like immunoreactive substance was located in the small vesicles with a diameter of about 50 nm, and that the HA positive vesicles accounted for more than 90% of the total vesicles, and the large vesicles (diameter 100 nm) and individual small vesicles were not H. A immunoreactive substances. The results showed that HA mainly existed in the vesicles of the sympathetic neurons. This result provides a direct morphological evidence for the definition of HA as a new sympathetic neurotransmitter at the subcellular level.
3. the kinetics of histamine synaptic vesicle circulation in SCG neurite outgrowth
Using FM1-43 fluorescent dye and HA immunofluorescence staining, the release kinetics of HA vesicles and the movement process of HA vesicles were observed on the SCG neurites in the cultured guinea pigs. The laser confocal microscope imaging and statistical analysis were made. The results of decolorization (Destaining) showed that the red fluorescence signal intensity of HA in the protuberance of the SCG God and the FM1-43 green in the protuberance of the SCG God. The intensity of the fluorescence signal increased with the increase of the depolarization time, and showed a gradual weakening. Both showed significant positive correlation (r = 0.989, P 0.01). It showed that the release of HA followed the general rule of the kinetics of vesicle release. The red fluorescence intensity and FM1-43 representing the HA were determined by the fixed cells at different time points of depolarization. The results of green fluorescence intensity indicate that the red fluorescence intensity of HA is stronger than the green fluorescence intensity of FM1-43. Due to the depolarization conditions used in this study, the FM1-43 fluorescence signal mainly reflects the release of circulating vesicles. The difference between the fluorescence intensity of HA and FM1-43 suggests that the vesicles stored on the HA at the nerve sites may exist. The results of Fluorescence recovery after photobleaching (FRAP) combined with immunohistochemical study showed that the diffusion rate of HA vesicles at SCG neurites was 0.09 u m2/s, and the other vesicles of 0.08 u m2/ s.HA vesicles were not significantly worse than other vesicles. Different.
Conclusion:
1. HA is ubiquitous in the sympathetic nervous system of many animals and coexists with the classical sympathetic neurotransmitter NE.
2. HA is mainly stored in the sympathetic synaptic vesicles.
The release of HA vesicles in 3. sympathetic neurons follows the classical neurotransmitter vesicle cycle dynamics. The storage of HA vesicles may be divided into release vesicles and reserve vesicles.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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7 童玲;左科;楊穎;楊小軍;錢偉;侯曉華;;去甲腎上腺素在REM睡眠剝奪降低大鼠內(nèi)臟感覺(jué)功能中的作用[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)消化病學(xué)術(shù)會(huì)議論文匯編（上冊(cè)）[C];2007年

8 唐文豪;閆龍濤;侯小飛;趙磊;王國(guó)良;黃毅;馬潞林;;后腹腔鏡左腎上腺區(qū)胃腸道外間質(zhì)瘤根治術(shù)[A];第十五屆全國(guó)泌尿外科學(xué)術(shù)會(huì)議論文集[C];2008年

9 陳慕雁;楊紅生;;應(yīng)激激素體外誘導(dǎo)對(duì)櫛孔扇貝血細(xì)胞活性的影響[A];中國(guó)動(dòng)物學(xué)會(huì)、中國(guó)海洋湖沼學(xué)會(huì)貝類學(xué)分會(huì)第八次會(huì)員代表大會(huì)暨第十三次全國(guó)貝類學(xué)術(shù)討論會(huì)論文摘要集[C];2007年

10 黃俊龍;周江睿;馬騫;蔣春雷;;去甲腎上腺素通過(guò)toll樣受體4促進(jìn)巨細(xì)胞炎癥因子的分泌[A];2008年神經(jīng)內(nèi)分泌暨神經(jīng)免疫內(nèi)分泌學(xué)術(shù)研討會(huì)論文摘要匯編[C];2008年

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2 葉忠孝;吃魚(yú)：請(qǐng)嚴(yán)防組胺中毒[N];醫(yī)藥養(yǎng)生保健報(bào);2006年

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5 鐘遠(yuǎn)超;組胺H_（2）受體拮抗劑有望避免神經(jīng)元的缺血損害[N];醫(yī)藥導(dǎo)報(bào);2004年

6 單曉聲;有些老人過(guò)年不能太激動(dòng)[N];醫(yī)藥養(yǎng)生保健報(bào);2009年

7 曉悅;吃蟹五忌[N];中國(guó)成人教育信息報(bào);2000年

8 劉國(guó)信;哪些魚(yú)類食不得[N];大眾衛(wèi)生報(bào);2000年

9 張曉穎;過(guò)敏性反應(yīng)病例分析（三）[N];農(nóng)村醫(yī)藥報(bào)（漢）;2007年

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2 耿爽;SOC通道在組胺誘導(dǎo)的人樹(shù)突狀細(xì)胞介導(dǎo)的Th_2反應(yīng)中的作用研究[D];武漢大學(xué);2012年

3 田爽;去甲腎上腺素促進(jìn)卵巢癌生長(zhǎng)和血管化的分子機(jī)制以及使用PTD4-t4EBP2逆轉(zhuǎn)化療抵抗的實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2009年

4 儲(chǔ)敏;Ciproxifan對(duì)實(shí)驗(yàn)動(dòng)物睡眠—覺(jué)醒的影響及其作用機(jī)制研究[D];復(fù)旦大學(xué);2004年

5 黃俊龍;去甲腎上腺素通過(guò)toll樣受體4促進(jìn)巨噬細(xì)胞炎癥因子的分泌[D];第二軍醫(yī)大學(xué);2008年

6 唐澤耀;肌球蛋白輕鏈的非Ca～（2+）依賴性磷酸化特征及其影響因素[D];大連醫(yī)科大學(xué);2005年

7 付作林;去甲腎上腺素在急性冬眠心肌中的釋放和作用[D];華中科技大學(xué);2006年

8 宋洪杰;皮膚組胺受體表達(dá)及西替利嗪的抗炎作用機(jī)制研究[D];第二軍醫(yī)大學(xué);2004年

9 陳�？�;藍(lán)斑對(duì)杏仁復(fù)合體神經(jīng)活動(dòng)的影響及在學(xué)習(xí)記憶中的作用[D];華東師范大學(xué);2007年

10 汪俊;神經(jīng)生長(zhǎng)因子啟動(dòng)哮喘腎上腺髓質(zhì)細(xì)胞冗余性致腎上腺素釋放障礙及蛋白質(zhì)組學(xué)初步研究[D];中南大學(xué);2007年

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1 胡榮;川芎揮發(fā)油解熱作用及機(jī)理研究[D];成都中醫(yī)藥大學(xué);2001年

2 肖榮;去甲腎上腺素對(duì)外周細(xì)胞炎性因子表達(dá)的影響[D];南華大學(xué);2007年

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4 冀永強(qiáng);大鼠腦組織中去甲腎上腺素及腎上腺素濃度變化與尿毒癥腦病的關(guān)系[D];青島大學(xué);2005年

5 蔡萌;去甲腎上腺素對(duì)THP-1巨噬細(xì)胞SR-AI和ABCA1表達(dá)的影響[D];南華大學(xué);2005年

6 石磊;音樂(lè)療法對(duì)化療期白血病患者生理與焦慮狀態(tài)影響的研究[D];吉林大學(xué);2007年

7 曹漫明;組胺聯(lián)合白細(xì)胞介素2激活自體造血干細(xì)胞移植物[D];第一軍醫(yī)大學(xué);2000年

8 陳伯成;納洛酮和Orexins對(duì)麻醉大鼠前額葉皮層神經(jīng)元電活動(dòng)的影響及其機(jī)制研究[D];第三軍醫(yī)大學(xué);2005年

9 薛麗;冰片對(duì)長(zhǎng)時(shí)連續(xù)作業(yè)大鼠覺(jué)醒能力損害干預(yù)的神經(jīng)機(jī)制[D];第三軍醫(yī)大學(xué);2006年

10 喬愛(ài)秀;發(fā)育中小鼠胃內(nèi)組胺免疫反應(yīng)細(xì)胞的形態(tài)學(xué)研究[D];山西醫(yī)科大學(xué);2003年

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