本文關(guān)鍵詞：中樞酸敏感離子通道對(duì)呼吸的調(diào)節(jié)及機(jī)制研究　出處：《復(fù)旦大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 酸敏感離子通道 呼吸調(diào)節(jié) 下丘腦外側(cè)區(qū) 延髓腹外側(cè)區(qū) 增食因子

【摘要】：目的：呼吸運(yùn)動(dòng)是一種受呼吸中樞調(diào)節(jié)的節(jié)律性的活動(dòng)。中樞化學(xué)感受器(central chemoreceptors)感受細(xì)胞外pH和CO2的濃度變化,將化學(xué)信號(hào)轉(zhuǎn)化電信號(hào),傳導(dǎo)給呼吸中樞的運(yùn)動(dòng)神經(jīng)元,完成對(duì)呼吸運(yùn)動(dòng)的調(diào)節(jié)。中樞化學(xué)感受器主要存在于延髓腹外側(cè)區(qū)(ventrolateral medulla, VLM),近幾年下丘腦外側(cè)區(qū)(lateral hypothalamus, LH)的Orexin神經(jīng)元在中樞化學(xué)感受中的作用也逐漸引起大家的關(guān)注。目前,中樞化學(xué)感受器感受細(xì)胞外H+濃度變化的離子通道機(jī)制還不明確,也一直是呼吸生理學(xué)研究的重點(diǎn)。1997年,Waldmann,R.克隆了第一個(gè)酸敏感離子通道(acid-sensing ion channels, ASICs),這是一類H+門控的陽(yáng)離子通道。研究發(fā)現(xiàn),ASICs參與機(jī)體許多生理和病理生理過(guò)程,如：學(xué)習(xí)記憶,痛覺、視覺、聽覺、味覺的感受,腦缺血損傷等,但是ASICs在呼吸的中樞調(diào)節(jié)中的作用卻知之甚少。本課題假設(shè)：1ASICs在VLM和LH都有表達(dá)并參與對(duì)呼吸的調(diào)節(jié)；2.LH的Orexin神經(jīng)元通過(guò)其胞膜上的ASICs通道感受細(xì)胞外pH值的變化,并調(diào)節(jié)呼吸運(yùn)動(dòng)。中樞的化學(xué)感受調(diào)節(jié)是快速調(diào)節(jié)維持機(jī)體酸堿平衡、內(nèi)環(huán)境穩(wěn)定的關(guān)鍵環(huán)節(jié)。中樞性呼吸衰竭、中樞性呼吸節(jié)律紊亂、先天性中樞性肺換氣不足綜合征等呼吸中樞調(diào)節(jié)紊亂的疾病對(duì)機(jī)體造成的危害是致命的,在 一些肺和呼吸道病變所引發(fā)的疾病如慢性阻塞性肺疾病、阻塞性睡眠呼吸暫停低通氣綜合征等疾病的病理過(guò)程中也伴有呼吸中樞調(diào)節(jié)功能的異常。因此,探討呼吸中樞調(diào)控機(jī)制不僅是呼吸生理學(xué)的理論突破,對(duì)呼吸系統(tǒng)疾病的病理過(guò)程研究和臨床預(yù)防與治療也具有重要的意義。 方法：1.實(shí)驗(yàn)用新生(1～5d)和成年(6～8w)SD大鼠,雌雄不計(jì),取延髓部位,用免疫組織化學(xué)ABC方法、免疫熒光雙標(biāo)、confoca1、westernblot和圖像分析技術(shù),觀察和比較ASIC1和ASIC2a亞單位在大鼠延髓和下丘腦的表達(dá),Orexin A神經(jīng)元在下丘腦的表達(dá),Orexin A神經(jīng)纖維在延髓的投射,Orexinl型受體(orexin type1receptor, OX1R)在延髓的表達(dá)以及ASIC1,ASIC2a亞單位與神經(jīng)纖維絲-H的共表達(dá)、ASIC1與ASIC2a亞單位的共表達(dá)、ASIC1與Orexin的共表達(dá)。2.成年(6～8w)SD大鼠腹腔麻醉后,應(yīng)用電生理實(shí)驗(yàn),采用腦立體定位和中樞核團(tuán)微量注射技術(shù),首先觀察在延髓腹外側(cè)區(qū)(VLM)和下丘腦外側(cè)區(qū)(LH)微量注射不同pH值的人工腦脊液對(duì)膈神經(jīng)放電活動(dòng)、血壓和心率的影響。然后在LH微量注射ASICs的非特異性阻斷劑Amiloride或ASICla選擇性阻斷劑PcTX1后,觀察酸化LH對(duì)呼吸和心血管系統(tǒng)的調(diào)節(jié)作用。觀察在側(cè)腦室微量注射Orexin A對(duì)膈神經(jīng)放電活動(dòng)、血壓和心率的影響。3.成年(6～8w)SD大鼠腹腔麻醉后,應(yīng)用電生理實(shí)驗(yàn),采用腦立體定位和中樞核團(tuán)微量注射技術(shù),在LH注射酸化人工腦脊液激活A(yù)SICs,同時(shí)在延髓的孤束核微量注射SB408124阻斷OX1R后,觀察膈神經(jīng)放電活動(dòng)、血壓、心率的變化。4.采用腦立體定位和中樞核團(tuán)微量注射技術(shù),成年(6-8w)SD大鼠的LH區(qū)微量注射Orexin-SAP特異性損毀該核團(tuán)的Orexin能神經(jīng)元,觀察大鼠攝食和體重的變化；應(yīng)用電生理實(shí)驗(yàn)觀察核團(tuán)損毀后,動(dòng)物的基礎(chǔ)呼吸頻率、血壓和心率的變化,以及酸化刺激LH區(qū)對(duì)心血管呼吸系統(tǒng)的調(diào)節(jié)。 結(jié)果 1. ASIC1和ASIC2a在成年和新生SD大鼠延髓的表達(dá)：(1)免疫組化的結(jié)果顯示：ASIC1和ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞在成年鼠的延髓有廣泛分布：ASIC1和ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞在第四腦室旁的中間核(intercalated nuclus of medulla, In)以及延髓背側(cè)的楔束副核(external cuneate nuclus, ECu)都有散在分布,在延髓的頭端腹外側(cè)區(qū)則有著廣泛分布。ASIC1和ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞在新生鼠延髓的表達(dá)略有不同：ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞主要分布在VLM區(qū),在延髓背側(cè)的ECu也有散在表達(dá)；ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞也主要分布在VLM區(qū),但是在延髓背側(cè)未觀察到ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞。在VLM區(qū),成年鼠組的ASIC2a免疫反應(yīng)的相對(duì)光密度(relative optical density, ROD)和陽(yáng)性細(xì)胞數(shù)值均低于新生鼠組的(p0.001,n=6)；成年鼠組的ASIC1免疫反應(yīng)的ROD值高于新生鼠組(p0.001,n=6)而細(xì)胞計(jì)數(shù)統(tǒng)計(jì)結(jié)果顯示成年鼠組的ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)少于新生鼠組(p0.001,n=6)。(2)應(yīng)用Western blot的方法：在成年鼠和新生鼠延髓都檢測(cè)到ASIC1和ASIC2a蛋白的表達(dá),ASIC1和ASIC2a在成年鼠組延髓的蛋白含量均低于新生鼠組(p0.01,p0.001,n=6)。(3)應(yīng)用免疫熒光雙染和免疫熒光共聚焦實(shí)驗(yàn)方法,在VLM區(qū)觀察到了ASIC1和ASIC2a與神經(jīng)纖維絲-H的共表達(dá),以及ASIC1和ASIC2a的共表達(dá)。 2. ASIC1和ASIC2a在成年SD大鼠下丘腦的表達(dá)：免疫組織化學(xué)實(shí)驗(yàn)結(jié)果顯示：ASIC1和ASIC2a兩個(gè)亞單位在成年SD大鼠的下丘腦廣泛表達(dá)：ASIC1和ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞在下丘腦背側(cè)區(qū)(dorsal hypothalamus area, DA)和下丘腦外側(cè)區(qū)(LH)都有分布,但是兩者的分布存在差異性。細(xì)胞計(jì)數(shù)結(jié)果顯示：LH區(qū)的ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)多于DA區(qū)(p0.001,n=7),ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)在DA區(qū)多于在LH的分布(p0.001,n=7)；在DA區(qū)ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)少于ASIC2a (p0.01,n=7),LH區(qū)ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)多于ASIC2a(p0.001,n=7)。對(duì)ASIC1和ASIC2a免疫反應(yīng)陽(yáng)性細(xì)胞的相對(duì)光密度(ROD)值的分析未發(fā)現(xiàn)兩者在DA區(qū)和LH區(qū)存在明顯差異。 3.在VLM區(qū)和LH區(qū)微量注射酸化人工腦脊液對(duì)呼吸及心血管系統(tǒng)的調(diào)節(jié)作用：(1)在SD大鼠的單側(cè)VLM區(qū)注射不同酸化程度的人工腦脊液(0.1μ1),pH值分別為pH7.4、6.5、6.0、5.5、5.0、4.5,與pH7.4組相比,觀察到pH6.5和pH6.0的人工腦脊液可以增加膈神經(jīng)放電強(qiáng)度(p0.05,n=12),對(duì)呼吸頻率、血壓、心率均沒有明顯的影響。(2)在SD大鼠的單側(cè)LH區(qū)注射不同酸化程度的人工腦脊液(0.1μ1),pH值分別為pH7.4、6.5、5.5、4.5,與pH7.4組相比,觀察到pH6.5的人工腦脊液可以明顯增大膈神經(jīng)放電強(qiáng)度(p0.001,n=6),對(duì)平均動(dòng)脈壓、心率和呼吸頻率沒有明顯影響。(3)在LH區(qū)微量注射ASICs非特異性阻斷劑Amiloride (10mM,0.1μl)或ASIC1a特異性阻斷劑PcTX1(10nM,0.1μl)之后立刻注射pH6.5人工腦脊液(0.1μ1),明顯阻斷了pH6.5人工腦脊液的增大膈神經(jīng)的效應(yīng)(p0.001,n=6),而單獨(dú)注射Amiloride或PcTX1對(duì)呼吸沒有明顯抑制作用。各個(gè)用藥組對(duì)呼吸頻率、血壓、心率沒有明顯的影響。 4. Orexin A對(duì)呼吸和心血管系統(tǒng)的調(diào)節(jié)作用：(1)免疫組化結(jié)果顯示：Orexin A免疫反應(yīng)陽(yáng)性纖維存在于延髓的孤束核(nucleus tractus solitary, NTS),并且在NTS也觀察到OX1R免疫反應(yīng)陽(yáng)性神經(jīng)元的存在。(2)單側(cè)側(cè)腦室微量注射不同濃度的Orexin A(10、100、10001μg/ml,5μl),劑量依賴性地增強(qiáng)膈神經(jīng)放電強(qiáng)度、升高動(dòng)脈血壓、加快心率,但對(duì)呼吸頻率沒有影響。三個(gè)濃度組的膈神經(jīng)放電強(qiáng)度分別與生理鹽水對(duì)照組相比都有統(tǒng)計(jì)意義(p0.05,p0.01,p0.001,n=6)。100、1000μg/ml兩個(gè)濃度組的平均動(dòng)脈壓與生理鹽水對(duì)照組相比有統(tǒng)計(jì)意義(p0.05,p0.01,n=6)。100、1000μg/ml濃度組的心率與生理鹽水對(duì)照組相比有統(tǒng)計(jì)意義(p0.01,p0.01,n=6)。 5. ASIC1與Orexin A在LH區(qū)的共表達(dá),以及它們?cè)诤粑{(diào)節(jié)中的相互作用：(1)應(yīng)用免疫組化實(shí)驗(yàn)在LH區(qū)觀察到Orexin A免疫反應(yīng)陽(yáng)性細(xì)胞。應(yīng)用免疫熒光雙染和免疫熒光共聚焦實(shí)驗(yàn)方法,在LH區(qū)觀察到ASIC1亞單位與Orexin A的共表達(dá)。(2)在單側(cè)LH區(qū)注射pH6.5人工腦脊液(0.1μl),同時(shí)在同側(cè)NTS注射SB408124(0.1μl)與注射pH7.4的人工腦脊液(0.1μl)相比,明顯阻斷了pH6.5人工腦脊液的增加呼吸強(qiáng)度的效應(yīng)(p0.01,n=6),而單側(cè)LH區(qū)注射pH7.4的人工腦脊液,同時(shí)同側(cè)NTS注射SB408124對(duì)呼吸強(qiáng)度沒有影響。(3)雙側(cè)LH區(qū)注射Orexin-SAP(0.43mg/ml,400nl/側(cè))兩周后,觀察到損毀組大鼠的體重明顯下降,應(yīng)用尼氏染色和免疫組化方法,觀察到兩周后LH區(qū)的Orexin A神經(jīng)元數(shù)量明顯減少,僅有少量殘存的Orexin A神經(jīng)元。應(yīng)用電生理的實(shí)驗(yàn)技術(shù),觀察到損毀組大鼠與Blank-SAP對(duì)照組相比平均動(dòng)脈壓下降,心率減慢(p0.01,n=6),呼吸頻率沒有變化。在損毀組大鼠的單側(cè)LH區(qū)注射pH6.5人工腦脊液,對(duì)膈神經(jīng)放電強(qiáng)度不再有興奮作用。 結(jié)論： 1.AISC1和ASIC2a亞單位在SD大鼠的延髓呼吸相關(guān)核團(tuán)VLM區(qū)和下丘腦的LH區(qū)都有表達(dá)。隨著發(fā)育成熟,ASIC1和ASIC2a亞單位蛋白表達(dá)量在成年鼠延髓的比在新生鼠減少,雖然ASIC1細(xì)胞數(shù)量減少,但是ASIC1免疫反應(yīng)陽(yáng)性細(xì)胞的相對(duì)光密度值(ROD)增加,提示成年鼠延髓單個(gè)細(xì)胞上的ASIC1表達(dá)增加。ASIC2a的ROD和細(xì)胞數(shù)量在成年鼠VLM區(qū)都比在新生鼠減少。ASICs亞單位表達(dá)量的變化可能與不同發(fā)育階段中樞對(duì)化學(xué)刺激的敏感性不同有關(guān)。ASIC1和ASIC2a在LH區(qū)都有表達(dá),但是以ASIC1亞單位為主。提示下丘腦的LH區(qū)可能是新發(fā)現(xiàn)的中樞化學(xué)敏感區(qū)。實(shí)驗(yàn)結(jié)果為探討ASICs在呼吸中樞調(diào)節(jié)中的作用提供了形態(tài)學(xué)基礎(chǔ)。 2.在延髓VLM區(qū)和下丘腦LH區(qū)適度的酸化刺激都可以增加呼吸強(qiáng)度。觀察到酸化LH區(qū)引起的呼吸興奮效應(yīng)更明顯,其有效刺激的酸化程度為pH6.5,與ASIC la的pHo.5相符。ASICs的非特異性阻斷劑Amiloride和ASIC1a的特異性阻斷劑PcTX1幾乎都可以完全阻斷酸化LH區(qū)引起的興奮呼吸的效應(yīng)。在生理pH值范圍內(nèi),單獨(dú)阻斷LH區(qū)的ASICs通道對(duì)呼吸并沒有抑制作用。提示ASICs不僅在LH區(qū)存在,并且感受局部的化學(xué)變化,然后調(diào)節(jié)呼吸活動(dòng),在此調(diào)節(jié)過(guò)程中ASIC1a亞單位起主要作用。 3. ASIC1在下丘腦LH區(qū)的Orexin能神經(jīng)元上表達(dá)。LH區(qū)的Orexin(?)神經(jīng)元損毀后,酸化刺激LH區(qū)不再有興奮呼吸的作用。提示LH區(qū)的Orexin(?)神經(jīng)元可以感受局部的化學(xué)變化,可能是一類新發(fā)現(xiàn)的中樞化學(xué)感受器。而ASICs在下丘腦對(duì)呼吸的調(diào)節(jié)作用正是通過(guò)Orexin(?)神經(jīng)元實(shí)現(xiàn)的。側(cè)腦室微量注射外源性的OrexinA可以增加呼吸強(qiáng)度,Orexin能神經(jīng)纖維和OX1R在延髓NTS有表達(dá)。提示Orexin有可能通過(guò)向NTS的投射發(fā)揮對(duì)呼吸的調(diào)節(jié)作用。在NTS阻斷OX1R可以抑制酸化LH區(qū)增加呼吸強(qiáng)度的效應(yīng),而單獨(dú)阻斷NTS的OX1R對(duì)呼吸活動(dòng)沒有影響。說(shuō)明酸化刺激LH區(qū),同時(shí)阻斷NTS的OX1R所產(chǎn)生的效應(yīng)并非兩種相反作用的相互抵消,進(jìn)一步說(shuō)明酸化LH區(qū)是通過(guò)興奮Orexin神經(jīng)元,釋放Orexin與延髓NTS的OX1R結(jié)合,發(fā)揮興奮呼吸的效應(yīng)。
[Abstract]:Objective: breathing is a central respiratory regulation of rhythmic activity. Central chemoreceptor (central chemoreceptors) feel the concentration changes of extracellular pH and CO2, the chemical signal into electrical signal, transmitted to the motor neurons of the respiratory center, complete adjustment of the respiratory motion. Central chemoreceptor mainly exists in the ventrolateral medulla (ventrolateral medulla, VLM), in recent years, the lateral hypothalamic area (lateral hypothalamus, LH) of Orexin neurons in central chemoreception in gradually attracted attention. At present, the central chemoreceptor ion channel mechanism of extracellular H+ concentration of the feeling is still not clear, has been the research respiratory physiology focuses on.1997, Waldmann, R. cloning of the first acid sensing ion channels (acid-sensing ion, channels, ASICs), which is a kind of H+ gated cation channel research. Found that ASICs participates in many physiological and pathological processes, such as learning and memory, pain, vision, hearing, taste feeling, cerebral ischemia injury, but the role of ASICs in regulation of respiration in the CNS is poorly understood. This research hypothesis: the expression of 1ASICs and involved in the regulation of respiration in VLM and LH; 2.LH Orexin neurons through ASICs channels, its cell membrane feel the change in extracellular pH and regulate respiratory movement. Central chemoreceptor regulation is rapidly adjust and maintain the acid-base balance of the body, the key link in stable environment. Central respiratory failure, central respiratory rhythm disorders, congenital central pulmonary damage hypoventilation syndrome and other diseases of the respiratory central regulation disorders can lead to fatal, in
Some caused lung and respiratory diseases such as chronic obstructive pulmonary disease, obstructive sleep apnea hypopnea syndrome and other pathological process of disease also accompanied by abnormal respiratory function. Therefore, to explore the central respiratory regulation mechanism is not only the theoretical breakthrough of respiratory physiology, also has an important significance to the study of pathology the process of respiratory system diseases and clinical prevention and treatment.
Methods: 1. new experiments (1 ~ 5d) and adult (6 ~ 8W) SD rats, regardless of male and female, and medullary parts, using immunohistochemical ABC method, immunofluorescence, confoca1, Westernblot and image analysis technique to observe the expression and comparison of ASIC1 and ASIC2a subunits in the rat hypothalamus and spinal cord. The Orexin A expression in the hypothalamus neurons, Orexin A nerve fibers in the medulla of the projection type Orexinl receptor (orexin, type1receptor, OX1R) in the medulla and expression of ASIC1, co expression of ASIC2a subunit of neurofilament -H, co expression of ASIC1 and ASIC2a subunit,.2. adult co expression of ASIC1 and Orexin (6 ~ 8W) SD rats after intraperitoneal anesthesia, application of electrophysiological experiments, using stereotaxic and nucleus microinjection technique, first observed in the ventrolateral medulla (VLM) and lateral hypothalamic area (LH) injection of artificial cerebrospinal fluid with different pH values on the diaphragm of God The discharge activity, blood pressure and heart rate. Then in the blocking of non-specific LH microinjection of ASICs inhibitor Amiloride or ASICla selective antagonist PcTX1, observe the acidification LH regulating effect on respiratory and cardiovascular systems. In the observation of intracerebroventricular injection of Orexin A on the discharges of phrenic nerve activity, effects of.3. on blood pressure and heart rate (adult 6 ~ 8W) SD rats after intraperitoneal anesthesia, application of electrophysiological experiments, using stereotaxic and nucleus microinjection technique, the activation of ASICs in LH injection acidizing artificial cerebrospinal fluid, while blocking OX1R in NTS microinjection of SB408124 medulla after phrenic nerve discharge activity, blood pressure, heart rate changes by.4. stereotaxic and nucleus microinjection technique (6-8W), adult SD rats LH microinjection of Orexin-SAP specific damage to the nuclei of Orexin neurons, and observe the changes of food intake and body weight of rats; Electrophysiological experiments were carried out to observe the changes of basal respiratory rate, blood pressure and heart rate, as well as the regulation of acidification stimulation on the cardiovascular system in LH area after nuclear damage.
Result
1. the expression of ASIC1 and ASIC2a in the medulla of rats in adult and newborn SD: (1) the immunohistochemical results showed that medullary ASIC1 and ASIC2a immunoreactive cells in adult rats are widely distributed: intermediate nuclear ASIC1 and ASIC2a immunoreactive cells in the fourth ventricle (intercalated nuclus of medulla, In) wedge beam and accessory nucleus of the dorsal medulla oblongata (external cuneate, nuclus, ECu) are scattered in the end of the ventrolateral of medulla are widely distributed and.ASIC1 expression of ASIC2a immunoreactive cells in neonatal rat medulla is slightly different: ASIC1 immunoreactive cells were mainly distributed in the VLM region in the back rostral ECu also has scattered expression; ASIC2a immunoreactive cells were mainly distributed in the VLM region, but was not observed in the dorsal medulla of ASIC2a immunoreactive cells in the VLM area, ASIC2a group of immune response in adult rats The relative optical density (relative optical, density, ROD) and positive cells were lower than the value of neonatal rats group (p0.001, n=6); ASIC1 immune response of adult rats ROD group is higher than the value of neonatal group (p0.001, n=6) and the statistical results of cell count shows the number of ASIC1 immunoreactive cells in adult rat group than in neonatal rats group (p0.001, n=6). (2) methods Western blot: in the adult rat and neonatal rat medulla were detected the expression of ASIC1 and ASIC2a protein, ASIC1 and ASIC2a in protein content in adult rat medulla were lower than that of neonatal group (P0.01, p0.001, n=6). (3) immunofluorescence double immunofluorescence staining and confocal experiment method, the co expression of ASIC1 and ASIC2a and neurofilament -H in VLM region was observed, and the co expression of ASIC1 and ASIC2a.
2. the expression of ASIC1 and ASIC2a in the hypothalamus of adult SD rats: immunohistochemical results showed that ASIC1 and ASIC2a two subunits are widely expressed in adult SD rat hypothalamus: ASIC1 and ASIC2a immunoreactive cells in the dorsal hypothalamic area (dorsal hypothalamus, area, DA) and lateral hypothalamic area (LH) are have distribution, but the distribution of differences. Cell counting results showed that the LH area is more than the number of ASIC1 immunoreactive cells (p0.001, n=7) DA, ASIC2a immunoreactive cell number in the DA region than in the distribution of LH (p0.001, n=7); in the DA area is less than the number of ASIC1 immunoreactive cells ASIC2a (P0.01, n=7), LH ASIC1 immunoreactive positive cells than ASIC2a (p0.001, n=7). The relative optical density of ASIC1 and ASIC2a immunoreactive cells (ROD) value analysis showed no obvious difference in both DA and LH regions ISO.
3. in the VLM area and LH area of microinjection of acidification artificial cerebrospinal fluid regulating effect on respiratory and cardiovascular system: (1) in the VLM area of different degree of acidification of unilateral injection of rat SD artificial cerebrospinal fluid (0.1 1), pH = pH7.4,6.5,6.0,5.5,5.0,4.5, compared with group pH7.4, pH6.5 and pH6.0 were observed in artificial cerebrospinal fluid can increase the strength of the phrenic nerve discharge (P0.05, n=12), blood pressure, heart rate of respiratory frequency, there is no effect. (2) in the LH area of different degree of acidification of unilateral injection of rat SD artificial cerebrospinal fluid (0.1 1), pH = pH7.4,6.5,5.5,4.5, compared with pH7.4 group, observed pH6.5 artificial cerebrospinal fluid can increase the strength of the phrenic nerve discharge (p0.001, n=6), the mean arterial pressure, heart rate and respiratory rate had no obvious effect. (3) in the LH region of nonspecific microinjection of ASICs inhibitor Amiloride (10mM, 0.1 L) or ASIC1a inhibitor PcT X1 (10nM, 0.1 L) immediately after pH6.5 injection of artificial cerebrospinal fluid (0.1 ~ 1), significantly blocked pH6.5 artificial cerebrospinal fluid increases phrenic nerve effect (p0.001, n=6), and a single injection of Amiloride or PcTX1 had no obvious inhibitory effect on respiration. Each drug group of respiratory frequency, blood pressure, heart rate is not obvious effect.
4. Orexin A on the regulation of respiratory and cardiovascular system: (1) immunohistochemistry results showed that NTS A Orexin immunoreactive fibers were found in the medulla oblongata (nucleus tractus, solitary, NTS), and in NTS were also observed in OX1R immunoreactive neurons. (2) unilateral intracerebroventricular injection different concentrations of Orexin A (1010010001 g/ml, 5 L), dose dependently enhanced phrenic nerve discharge intensity increased, arterial blood pressure, heart rate and respiratory rate. But there is no effect on phrenic nerve discharge intensity of three concentration group respectively with saline control group has statistical significance compared (P0.05, P0.01 p0.001, n=6,.1001000) g/ml two concentration group the mean arterial pressure and saline control group had statistical significance (P0.05, P0.01, n=6 compared with saline.1001000) heart rate g/ml concentrations were statistically significant compared with the control group (P0.01, P0.01, n =6).
Co expression of 5. ASIC1 and Orexin A in the LH region, and their interactions in the regulation of respiration: (1) immunohistochemical experiment showed that A immunoreactive positive cells in the Orexin area of LH. Using immunofluorescence double staining and confocal immunofluorescence experiments, in the zone of LH co expression of ASIC1 the unit and the Orexin A. (2) in the LH region of pH6.5 injection unilateral artificial cerebrospinal fluid (0.1 L), at the same time on the same side NTS injection of SB408124 (0.1 L) and pH7.4 injection of artificial cerebrospinal fluid (0.1 L), significantly blocked the effect of increasing the respiratory intensity of pH6.5 artificial cerebrospinal fluid (P0.01, n=6), and unilateral LH injection in pH7.4 artificial cerebrospinal fluid, and ipsilateral NTS SB408124 injection had no effect on respiration. (3) bilateral LH injection of Orexin-SAP (0.43mg/ml, 400nl/ side) after two weeks, observed damage rats body weight decreased significantly, using Nissl staining and immunohistochemical method, the concept of Orexin observed that the number of A neurons in area LH decreased obviously after two weeks, only a small amount of residual Orexin A neurons. The application of experimental techniques of electrophysiology, observed damaged rats compared with Blank-SAP control group mean arterial pressure, heart rate (P0.01, n=6), respiratory rate did not change. In the lesioned group in the LH region of pH6.5 injection unilateral artificial cerebrospinal fluid, no longer have excitatory effects on phrenic nerve discharge intensity.
Conclusion:
The 1.AISC1 and ASIC2a subunits in SD rat medullary respiratory related nucleus VLM and LH in the hypothalamus region are expressed. With the maturation of ASIC1 and ASIC2a subunit protein expression in neonatal rats than in adult rat medulla decreased, while the number of ASIC1 cells decreased, but the relative optical density of ASIC1 immunoreactive cells the value (ROD) increased, suggesting that adult rat medullary mononuclear cells on the expression of ASIC1 increased the number of.ASIC2a and ROD cells in the adult rat VLM area than the reduction of.ASICs subunit expression may be related to different developmental stages of the central chemical stimuli sensitivity of different.ASIC1 and ASIC2a were expressed in the LH District in the newborn rat, but with ASIC1 subunit. The hypothalamic LH region may be found in central chemosensitive areas. The experimental results provide a morphological basis for the role of ASICs in the regulation of the respiratory center.
2. in the medulla VLM and hypothalamic LH area moderate acidification can increase the respiration intensity of stimulation. The observed respiratory stimulant effect caused by acidification LH region is more obvious, the degree of acidification of effective stimulus for pH6.5, ASIC and La pHo.5 are nonspecific.ASICs specific blocking agent Amiloride and ASIC1a the effect of blocking agent PcTX1 almost can be completely blocked by acidification induced by LH region excited breathing. In the range of physiological pH, single ASICs channel blocking LH area and had no inhibitory effect on respiration. It suggests that ASICs has not only in the LH region, and feel the local chemical changes, and then adjust the respiratory activity, during the adjusting process of ASIC1a subunit plays a major role.
3. ASIC1 in the hypothalamic LH area Orexin expression of neurons in.LH Orexin (?) neurons damage after acidification stimulated LH area no longer have excited breathing effect. LH region of Orexin (?) neurons can feel local chemical changes, may be a newly discovered class of central chemoreceptor and regulation. The effects of ASICs on respiration in the hypothalamus is through Orexin (?) neurons to achieve. Intracerebroventricular injection of exogenous OrexinA can increase the respiration intensity, Orexin nerve fibers and OX1R in medullary NTS

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R332

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