本文選題：N-甲基-D-天門(mén)冬氨酸 + 急性肺損傷�。� 參考：《中南大學(xué)》2006年博士論文

【摘要】： 第一章NMDA受體激活引起小鼠急性肺損傷 研究背景 N-甲基-D-天門(mén)冬氨酸(NMDA)受體是谷氨酸受體的重要亞型。NMDA受體過(guò)度激活引起的興奮性神經(jīng)毒性是引起神經(jīng)細(xì)胞損傷的最后共同通路，在多種急慢性腦損傷的發(fā)生中起有重要作用。近年來(lái)發(fā)現(xiàn)NMDA受體在肺臟也有較高水平的表達(dá)，但生物學(xué)意義尚不清楚。 急性呼吸窘迫綜合征(ARDS)是臨床常見(jiàn)危重疾病，發(fā)病機(jī)制尚未完全闡明。肺內(nèi)中性粒細(xì)胞的滯留和肺內(nèi)中性粒細(xì)胞、巨噬細(xì)胞的激活在ARDS的發(fā)生發(fā)展中起關(guān)鍵性作用。中性粒細(xì)胞和巨噬細(xì)胞可釋放谷氨酸。膿毒癥時(shí)肺靜脈中谷氨酸濃度明顯高于肺動(dòng)脈，提示在ARDS等疾病狀態(tài)下，肺組織中的NMDA受體有可能被過(guò)度激活。Said等人發(fā)現(xiàn)NMDA可引起離體灌流肺發(fā)生高通透性肺水腫。本室前期工作觀(guān)察到NMDA受體阻斷劑MK-801對(duì)腹腔注射谷氨酸引起的小鼠急性肺損傷具有明顯的保護(hù)作用，提示在器官水平和整體水平，NMDA受體激活可引起肺損傷。在整體水平研究NMDA受體激活后對(duì)肺組織的影響及可能的作用機(jī)制，將為深入研究肺臟等外周組織中NMDA受體的病理生理意義，拓展肺部疾病的防治研究提供新的思路。 方法 1．測(cè)量肺濕重和干重比值(W／D)并觀(guān)察組織病理變化，以反映肺組織的損傷程度。 2．生化試劑盒測(cè)定肺組織髓過(guò)氧化物酶(MPO)活性的改變以反映肺內(nèi)中性粒細(xì)胞的浸潤(rùn)程度。 3．尾靜脈取血，記數(shù)中性粒細(xì)胞百分比，反映外周血中性粒細(xì)胞的數(shù)目。 4．Western blot方法測(cè)定肺組織proSP-C和CCTα蛋白水平，反映肺泡Ⅱ型上皮細(xì)胞的功能。 結(jié)果 1．NMDA(50 mg／kg，ip)可引起肺組織W／D增加，病理切片炎癥滲出明顯，，NMDA受體的阻斷劑MK-801(0.1 mg／kg，ip)預(yù)處理30 min可明顯減輕由于NMDA引起的炎癥滲出。 2．NMDA(50 mg／kg，ip)能引起肺組織MPO活性增加，MK-801可抑制NMDA引起的MPO活性變化。 3．長(zhǎng)春新堿(5 mg／kg，iv)預(yù)處理4d可引起昆明小鼠外周血白細(xì)胞總數(shù)、中性粒細(xì)胞總數(shù)和中性粒細(xì)胞百分比的明顯減少，對(duì)NMDA引起的肺組織W／D的升高有明顯的抑制作用。 3．NMDA(50 mg／kg，ip)引起肺組織proSP-C和CCTα蛋白水平下降，NMDA受體阻斷劑MK-801可緩解NMDA對(duì)proSP-C和CCTα的影響。 結(jié)論 在整體條件下，NMDA可引起急性肺損傷，其機(jī)制與中性粒細(xì)胞的募集和肺泡Ⅱ型上皮細(xì)胞的功能障礙有關(guān)。 第二章NMDA受體在肺泡Ⅱ型上皮細(xì)胞的表達(dá) 研究背景 第一部分研究首次在整體水平證實(shí)NMDA受體的激活具有肺毒性效應(yīng)，但其肺毒性機(jī)制未完全闡明。肺泡Ⅱ型上皮細(xì)胞(ATⅡ)具有合成分泌表面活性物質(zhì)(PS)的特性，是維持肺泡正常功能和結(jié)構(gòu)的重要細(xì)胞。表面活性物質(zhì)結(jié)合蛋白-C的前體蛋白proSP-C僅存在于ATⅡ中，是ATⅡ的特征性蛋白。CTP：磷酸膽堿二胞苷酰基轉(zhuǎn)移酶α(CCTα)是合成PS主要脂質(zhì)成分磷脂酰膽堿(PC)的關(guān)鍵酶。腹腔注射N(xiāo)MDA可以使肺組織proSP-C和CCTα表達(dá)減少，表明肺組織合成PS脂質(zhì)成分和蛋白成分的能力下降，提示ATⅡ受損。但NMDA是直接對(duì)ATⅡ發(fā)揮損傷作用，還是通過(guò)作用于其他細(xì)胞啟動(dòng)損傷效應(yīng)、釋放損傷性介質(zhì)從而間接導(dǎo)致ATⅡ的損害，尚有待于進(jìn)一步的探索。 NR1是構(gòu)成NMDA受體的必需組份。研究表明在肺組織的外周、中段、主干均有NR1的mRNA及蛋白的表達(dá)。放射配基結(jié)合實(shí)驗(yàn)也證實(shí)在肺泡壁上有NMDA受體表達(dá)，但在ATⅡ細(xì)胞，NMDA受體有無(wú)表達(dá)尚無(wú)直接的證據(jù)。 方法 1．免疫組織化學(xué)(SABC法和雙標(biāo)記法)技術(shù)檢測(cè)在人肺組織ATⅡ細(xì)胞上NR1的表達(dá)。 2．原代分離的大鼠ATⅡ細(xì)胞經(jīng)免疫黏附法純化2次后，用堿性磷酸酶(AKP)染色鑒定純度。 3．RT-PCR法和Western blot分別檢測(cè)ATⅡ細(xì)胞上NR1的mRNA和蛋白分子。免疫細(xì)胞化學(xué)觀(guān)察ATⅡ細(xì)胞上NR1的表達(dá)。 結(jié)果 正常人肺組織石蠟切片免疫組化結(jié)果顯示ATⅡ細(xì)胞上有NR1的表達(dá)。 原代分離純化的ATⅡ細(xì)胞經(jīng)AKP染色鑒定純度為90％以上，可檢測(cè)到NR1的mRNA和蛋白分子，免疫細(xì)胞化學(xué)方法顯示ATⅡ細(xì)胞呈NR1陽(yáng)性。 結(jié)論 ATⅡ細(xì)胞有NMDA受體的NR1亞單位表達(dá)。 第三章NMDA對(duì)肺表面活性物質(zhì)合成的影響 研究背景 肺泡Ⅱ型上皮細(xì)胞(ATⅡ)是肺泡上皮的主要組成細(xì)胞，可以合成分泌肺表面活性物質(zhì)(PS)。ATⅡ細(xì)胞或PS的受損在ARDS、肺纖維化多種肺部疾病的發(fā)生發(fā)展中起著重要作用。 磷脂酰膽堿(PC)是PS中脂質(zhì)的主要成分。CTP：磷酸膽堿二胞苷酰基轉(zhuǎn)移酶(CCTα)是PC合成的限速酶，也是PC生物合成的重要調(diào)控部位。 第三部分的研究發(fā)現(xiàn)ATⅡ有NMDA受體表達(dá)。前期研究已經(jīng)在整體水平證實(shí)NMDA受體的激活可導(dǎo)致肺組織CCTα蛋白水平下降；在組織培養(yǎng)水平證實(shí)NMDA受體的激活可降低肺組織CCTαmRNA的水平，抑制肺組織PC生物合成；但是在細(xì)胞水平NMDA可否直接影響PS的合成仍有待于進(jìn)一步的研究。 方法 液閃記數(shù)測(cè)定[~3H]氯化膽堿的摻入量，反映PC合成的變化。 RT-PCR和Western blot分別測(cè)定CCTα的mRNA水平和微粒體中的CCTα蛋白水平，揭示PC合成變化的生物化學(xué)機(jī)制。 Real time PCR測(cè)定CCTα和SP-A、SP-B、SP-C、SP-D的mRNA水平，反映CCTα和SPs轉(zhuǎn)錄水平的變化。 結(jié)果 濃度為30μmol／L～100μmol／L的NMDA處理12 h，可以劑量依賴(lài)方式抑制A549細(xì)胞[~3H]氯化膽堿的摻入量。 10μmol／L～1000μmol／L的NMDA處理12 h可以劑量依賴(lài)方式降低A549細(xì)胞CCTα的mRNA水平和微粒體中的CCTα蛋白水平。100μmol／L的NMDA可以引起ATⅡ細(xì)胞CCTα的mRNA水平降低。 100μmol／L的NMDA處理12 h可以引起ATⅡ細(xì)胞SP-B、SP-C、SP-D的mRNA水平降低，而對(duì)SP-A mRNA的影響不明顯。 結(jié)論 NMDA可通過(guò)減少CCTα的轉(zhuǎn)錄，降低高活性的CCTα蛋白水平，導(dǎo)致PC合成減少。NMDA對(duì)SP-B、SP-C、SP-D的轉(zhuǎn)錄也有抑制作用。 第四章NMDA抑制CTP：磷酸膽堿二胞苷�；D(zhuǎn)移酶表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)通路的研究 研究背景 肺泡Ⅱ型上皮細(xì)胞(ATⅡ)合成分泌的肺表面活性物質(zhì)(PS)是維持正常呼吸功能不可缺少的生物活性物質(zhì)。CTP：磷酸膽堿二胞苷�；D(zhuǎn)移酶α(CCTα)是PS主要脂質(zhì)成分磷脂酰膽堿(PC)合成的限速酶。 在CCTα基因近端啟動(dòng)子存在核因子κB(NFκB)的結(jié)合位點(diǎn)，但NFκB對(duì)CCTα轉(zhuǎn)錄有何影響未見(jiàn)報(bào)道。神經(jīng)細(xì)胞在受到興奮性毒性損傷時(shí)核內(nèi)NFκB活性增強(qiáng)，在此過(guò)程中一氧化氮合酶(NOS)活化是NFκB激活的上游事件。在整體實(shí)驗(yàn)和離體灌流肺模型的研究表明，NOS的激活是導(dǎo)致NMDA肺組織損傷的重要環(huán)節(jié)。本室在細(xì)胞水平的研究中發(fā)現(xiàn)NMDA可以抑制ATⅡ細(xì)胞CCTα的轉(zhuǎn)錄，推測(cè)在該效應(yīng)中，與興奮性神經(jīng)毒性相似，NMDA激活NOS并由此導(dǎo)致NFκB活化，從而調(diào)節(jié)CCTα的轉(zhuǎn)錄。 方法 Western blot測(cè)定細(xì)胞核中P65蛋白水平的變化，反映NFκB的激活；測(cè)定CCTα蛋白水平，反映CCTα的表達(dá)變化。 生化試劑盒測(cè)定細(xì)胞中一氧化氮合酶(NOS)活性和釋放到培養(yǎng)液中的一氧化氮(NO)量；Western blot檢測(cè)NOS阻斷劑L-NNA預(yù)處理對(duì)細(xì)胞核中P65蛋白水平的影響，反映NOS在NFκB活化過(guò)程中的作用。 結(jié)果 NMDA(100μmol／L)處理2 h，8 h均檢測(cè)到A549細(xì)胞核中P65蛋白增加。 NFκB的核轉(zhuǎn)位抑制劑SN-50(360μmol／L)預(yù)處理2 h可明顯抑制NMDA(100μmol／L)所致的A549細(xì)胞核中P65的增加，并對(duì)CCTα蛋白的減少具有明顯的保護(hù)作用。 NMDA(100μmol／L)處理2 h，A549細(xì)胞中NOS的活性和釋放到培養(yǎng)液中的NO無(wú)明顯變化；而處理7 h則可使A549細(xì)胞中NOS的活性升高，NO釋放量增多。 L-NNA(2.5 m mol／L)預(yù)處理3 h可明顯抑制NMDA(100μmol／L)處理后8 h A549細(xì)胞核中P65的增加。 結(jié)論 NMDA通過(guò)激活NFκB抑制CCTα的表達(dá)，NOS活化是激活NFκB的上游途徑。
[Abstract]:Chapter 1 activation of NMDA receptor causes acute lung injury in mice
Research background
N- methyl -D- aspartic acid (NMDA) receptor, an important subtype of glutamate receptor, is an important subtype of.NMDA receptor excitability induced neurotoxicity, the final common pathway that causes nerve cell damage, and plays an important role in the occurrence of a variety of acute and chronic brain damage. In recent years, it has been found that NMDA receptors have high levels of expression in the lungs, but they have been produced in the lung. The significance of physical science is not yet clear.
Acute respiratory distress syndrome (ARDS) is a common critical disease in the clinic. The pathogenesis is not fully elucidated. The retention of neutrophils in the lung and the activation of neutrophils and macrophages in the lung play a key role in the development of ARDS. Neutrophils and macrophages can release glutamate. The degree of the pulmonary artery was significantly higher than that of the pulmonary artery. It is suggested that the NMDA receptor in the lung tissue may be excessively activated by.Said and others in the condition of ARDS and other diseases. It is found that NMDA can cause high permeability pulmonary edema in the isolated perfusion lung. The early work of this room has observed that the NMDA receptor blocker MK-801 is evident in the acute lung injury induced by the intraperitoneal injection of glutaric acid in mice. The protective effect of NMDA receptor activates lung injury at organ level and overall level. The study of the effect and possible mechanism of NMDA receptor activation on lung tissue at the whole level will provide a new idea for the in-depth study of the pathophysiological significance of NMDA receptors in the peripheral tissue of the lung and the prevention and treatment of pulmonary diseases.
Method
1. to measure lung wet weight and dry weight ratio (W / D) and observe histopathological changes to reflect the degree of lung tissue injury.
2. biochemical assay kit was used to measure the activity of myeloperoxidase (MPO) in lung tissue to reflect the infiltration of neutrophils in the lung.
3. tail venous blood was taken, counting the percentage of neutrophils, reflecting the number of neutrophils in peripheral blood.
4.Western blot method was used to detect proSP-C and CCT alpha protein levels in lung tissue, reflecting the function of alveolar type II epithelial cells.
Result
1.NMDA (50 mg / kg, IP) can cause the increase of W / D in lung tissue, and the inflammatory exudation of pathological section is obvious. The pretreatment of NMDA receptor blocker MK-801 (0.1 mg / kg, IP) can obviously reduce the inflammatory exudation caused by the 30 min.
2.NMDA (50 mg / kg, IP) could increase the activity of MPO in lung tissue, and MK-801 could inhibit the change of MPO activity induced by NMDA.
3. vincristine (5 mg / kg, IV) pretreated 4D could cause the total number of white blood cells in the peripheral blood of Kunming mice, the total number of neutrophils and the percentage of neutrophils decreased significantly, which had obvious inhibitory effect on the increase of W / D in the lung tissue caused by NMDA.
3.NMDA (50 mg / kg, IP) reduced the level of proSP-C and CCT alpha protein in lung tissue, and NMDA receptor blocker MK-801 could alleviate the effect of NMDA on proSP-C and CCT alpha.
conclusion
Under the overall conditions, NMDA can cause acute lung injury, which is related to the recruitment of neutrophils and dysfunction of alveolar type II epithelial cells.
The second chapter is the expression of NMDA receptor in alveolar type II epithelial cells.
Research background
The first part of the study showed that the activation of NMDA receptor had lung toxicity for the first time, but the mechanism of lung toxicity was not fully elucidated. The pulmonary alveolar type II epithelial cells (AT II) had the characteristics of synthetic and secretory surfactant (PS), an important cell to maintain normal function and structure of the alveoli. The precursor of surfactant binding protein -C Protein proSP-C only exists in AT II and is the characteristic protein.CTP of AT II: phosphachylcholine two cytidine acyltransferase alpha (CCT alpha) is a key enzyme in the synthesis of PS major lipid component phosphatidylcholine (PC). Intraperitoneal injection of NMDA can reduce the expression of proSP-C and CCT alpha in lung tissue, indicating the ability of lung tissue to synthesize PS lipid and protein components. Decrease, suggesting that AT II is damaged. But NMDA is a direct damage to AT II, or by acting on the damage effect of other cells, releasing damaging media and indirectly causing the damage of AT II. It still remains to be further explored.
NR1 is an essential component of the NMDA receptor. The study shows that there are NR1 mRNA and protein expressions in the peripheral, middle, and main arteries of the lung tissue. The radioligand binding assay also confirmed the expression of NMDA receptor on the alveolar wall, but there is no direct evidence for the expression of NMDA receptor in AT II cells.
Method
1. immunohistochemical method (SABC and double labelling) was used to detect the expression of NR1 in human lung AT II cells.
2. the purified rat AT II cells were purified by immunological adhesion for 2 times, and their purity was identified by alkaline phosphatase (AKP) staining.
3.RT-PCR and Western blot were used to detect the mRNA and protein molecules of NR1 on AT II cells. The expression of NR1 on AT II cells was observed by immunocytochemistry.
Result
Immunohistochemical staining of paraffin sections of normal lung tissue showed NR1 expression on AT II cells.
The purity of the original purified AT II cells was more than 90% by AKP staining, and the mRNA and protein molecules of NR1 were detected. The immunocytochemical method showed that AT II cells were NR1 positive.
conclusion
AT II cells have the expression of NR1 subunit of the NMDA receptor.
The third chapter is about the effect of NMDA on the synthesis of pulmonary surfactant.
Research background
Alveolar type II epithelial cells (AT II) are the main constituent cells of alveolar epithelium. The damage of PS.AT II cells or PS can be synthesized in ARDS, and it plays an important role in the development and development of pulmonary fibrosis of various pulmonary diseases.
Phosphatidylcholine (PC) is the main component of lipid in PS,.CTP: phosphocholine two cytidine acyltransferase (CCT alpha) is a speed limiting enzyme in PC synthesis, and is also an important regulatory part of PC biosynthesis.
The third part of the study found that AT II has NMDA receptor expression. Earlier studies have confirmed that the activation of NMDA receptor can lead to a decrease in the level of CCT alpha protein in lung tissue at the overall level; at the level of tissue culture, the activation of NMDA receptor can reduce the level of CCT alpha mRNA in lung tissue and inhibit PC biosynthesis in lung tissue, but at the cell level NMDA can be found. No direct impact on the synthesis of PS remains to be further studied.
Method
The amount of choline chloride in [~3H] was determined by liquid scintillation counting, which reflected the change of PC synthesis.
RT-PCR and Western blot were used to measure the mRNA level of CCT alpha and the CCT alpha protein level in microsomes, revealing the biochemical mechanism of PC synthesis.
Real time PCR measured the mRNA level of CCT alpha and SP-A, SP-B, SP-C, SP-D, reflecting the change of CCT alpha and SPs transcriptional level.
Result
The NMDA concentration of 30 mol / L to 100 mol / L treated with 12 h could inhibit the incorporation of [~3H] choline chloride in A549 cells in a dose-dependent manner.
The NMDA treatment of 10 mol / L ~ 1000 mu mol / L can reduce the mRNA level of CCT alpha in A549 cells in a dose-dependent manner and CCT alpha protein level in microsomes.
The treatment of 12 h with 100 mol / L NMDA could cause the mRNA level of SP-B, SP-C and SP-D in AT II cells to decrease, but the effect on SP-A mRNA was not obvious.
conclusion
NMDA can reduce the transcription of CCT alpha and reduce the level of high activity CCT alpha protein, resulting in decreased PC synthesis..NMDA also inhibits SP-B, SP-C and SP-D transcription.
Fourth chapter NMDA inhibits CTP: signal transduction pathway of phosphorylcholine two cytidine acyltransferase expression.
Research background
Pulmonary surfactant (PS), which is synthesized and secreted from alveolar type II epithelial cells (AT II), is an essential bioactive substance to maintain normal respiratory function,.CTP: phosphocholine two cytidine acyltransferase alpha (CCT alpha) is a limiting enzyme in the synthesis of phosphatidylcholine (PC), the main lipid component of PS.
There is a binding site of nuclear factor kappa B (NF kappa B) in the proximal promoter of the CCT alpha gene, but there is no report on the effect of NF kappa B on the transcription of CCT a. The activation of NF kappa B in the nucleus is enhanced during excitotoxicity damage. During this process, the activation of nitric oxide synthase (NOS) is the upstream event of NF kappa B excitability. In the whole experiment and in vitro perfusion lung model The study showed that activation of NOS was an important link in the injury of NMDA lung tissue. In the cell level study, it was found that NMDA could inhibit the transcription of CCT alpha in AT II cells. In this effect, it is similar to excitatory neurotoxicity. NMDA activates NOS and thus leads to the activation of NF kappa B, thus regulating the transcription of CCT alpha.
Method
Western blot was used to measure the level of P65 protein in the nucleus, reflecting the activation of NF kappa B, and measuring the level of CCT alpha protein, reflecting the change of CCT alpha expression.
The activity of nitric oxide synthase (NOS) in cells and the amount of nitric oxide (NO) released into the culture medium were measured by biochemical kits, and the effect of NOS blocker L-NNA preconditioning on the level of P65 protein in the nucleus was detected by Western blot, reflecting the role of NOS in the activation of NF kappa B.
Result
NMDA (100 mol / L) treated 2 h, and 8 h detected A549 protein increased in the nucleus of A549.
The pretreatment of NF kappa B nuclear transposition inhibitor SN-50 (360 Mu mol / L) pretreated 2 h could obviously inhibit the P65 increase in A549 nuclei induced by NMDA (100 u mol / L), and had a significant protective effect on the reduction of CCT alpha protein.
NMDA (100 mu mol / L) treated 2 h. The activity of NOS in A549 cells and the NO released into the culture medium did not change obviously, while the treatment of 7 h could increase the activity of NOS in A549 cells and increase the NO release.
Pretreatment of 3 h with L-NNA (2.5 m mol / L) significantly inhibited the increase of P65 in 8 h A549 nuclei after NMDA (100 mol mol / L) treatment.
conclusion
NMDA inhibits CCT alpha expression by activating NF kappa B, and NOS activation is the upstream pathway to activate NF kappa B.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 尚利宏;NMDA受體激活對(duì)肺泡巨噬細(xì)胞分泌作用的影響及機(jī)制研究[D];中南大學(xué);2008年

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1 史小娟;NMDA受體激活在急性肺損傷CCTα表達(dá)下調(diào)中的作用及機(jī)制[D];中南大學(xué);2008年

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