本文選題：氧化應激損傷 + 海馬神經(jīng)元　； 參考：《瀘州醫(yī)學院》2014年碩士論文

【摘要】：研究背景 血紅素加氧酶(heme oxygenase, HO)是血紅素分解代謝過程中的限速酶，人體內(nèi)的CO主要是由血紅素加氧酶(HO)代謝產(chǎn)生。HO有三種類型：誘導型(HO-1)、組成型(HO-2)及尚未明確的HO-3[1]。研究表明，HO-1不僅在機體生理狀態(tài)下發(fā)揮作用，更主要是在機體其他非正常狀態(tài)/應激狀態(tài)下發(fā)揮作用。HO-1參與活性氧、活性氮、缺血、細菌脂多糖、血紅素等誘導的氧化應激反應。研究顯示HO-1表達升高可減輕應激損傷大鼠的腦組織形態(tài)與氧化應激損傷程度，其機制可能與Nrf2-Keap1［(NF-E2-relatedfactor2,轉(zhuǎn)錄因子NF-E2相關(guān)因子2)-(Kelch-likeECH－associated protein1,胞漿蛋白伴侶分子)］和PI3K/Akt/GSK-3β(phosphatidyllinositol-3-kinase/Akt/glycogen syntheses kinase-3β,磷脂酰肌醇激酶-3/絲蘇氨酸蛋白激酶/糖原合成酶激酶-3β)通路有關(guān)[2-3]。血紅素在HO-1作用下分解為膽紅素、游離鐵離子和CO，這些物質(zhì)參與自由基的清除、炎癥因子的抑制釋放、轉(zhuǎn)錄激活因子的上調(diào)、tau蛋白的磷酸化和抑制HO-1的泛素化等病理生理過程[4-7]。HO-2主要分布在中樞神經(jīng)系統(tǒng)及睪丸，它所產(chǎn)生的CO在神經(jīng)信號傳遞中起重要作用，與CO發(fā)揮神經(jīng)遞質(zhì)的作用密切相關(guān)；HO-2維持腦的正常生理功能，而HO-1與神經(jīng)系統(tǒng)疾病密切相關(guān)。 一氧化氮合酶(nitric oxide syntheses, NOS)有三種同工酶，包括主要存在于神經(jīng)系統(tǒng)中的神經(jīng)型一氧化氮合酶(nitric oxide syntheses, nNOS)，存在于巨噬細胞、肝細胞和神經(jīng)膠質(zhì)細胞中的誘導型一氧化氮合酶(nitric oxidesyntheses, eNOS)和主要存在于內(nèi)皮細胞中的內(nèi)皮型一氧化氮合酶(nitricoxide syntheses, iNOS)。在生物體內(nèi)，NOS利用L-精氨酸和分子氧作為底物，NADPH輔酶作為輔助因子，經(jīng)過一系列氧化反應生成一氧化氮(NO)和瓜氨酸。在神經(jīng)系統(tǒng)中NO作為一種重要的信號分子，參與了學習與記憶等重要的神經(jīng)生理活動，同時對腦部血流具有調(diào)節(jié)作用，并參與神經(jīng)系統(tǒng)的免疫防御。另一方面過量的NO又生產(chǎn)細胞毒性氧化物質(zhì)(如氮自由基、過氧亞硝酸鹽等)，與腦缺血損傷、癡呆、帕金森病等疾病的發(fā)生、發(fā)展有密切關(guān)系。因此，在正常生理條件下，神經(jīng)系統(tǒng)中存在著從時間與空間上精確調(diào)控NO產(chǎn)生、釋放、擴散與滅活的機制，而這主要是通過調(diào)控nNOS的活化與去活化實現(xiàn)的。研究證明nNOS通過與CB2(type2cannabinoidreceptor,大麻素受體2)結(jié)合直接調(diào)節(jié)nNOS，進而與抗氧化蛋白Hsp70和抗凋亡蛋白Bcl-2共同完成CB2通路，，延遲神經(jīng)退行性變[8]。一氧化氮合酶調(diào)節(jié)體內(nèi)一氧化氮自由基和氧自由基平衡[9]，參與腦創(chuàng)傷后損傷及神經(jīng)再生、缺血再灌注損傷、神經(jīng)系統(tǒng)退行性疾病、腦卒中、精神分裂性疾病等病理生理過程[10-13]，其機制與脂質(zhì)過氧化、蛋白質(zhì)羰基化、蛋白硝基化和自由基清除有關(guān)[14-15]。Ríos R等[16]探討低濃度砷中毒性大鼠中樞損害模型發(fā)現(xiàn)，不僅活性氧與脂質(zhì)過氧化產(chǎn)物可作為氧化應激損傷的生物學標志，nNOS也可作為氧化應激損傷的生物學標志之一。 姜黃素(curcumin, Cur)是姜科植物Curcuma longa根莖中分離得到的一種脂溶性多酚類化合物，具有抗氧化、抗炎癥、抗腫瘤、抗纖維化、保護心血管系統(tǒng)和消化系統(tǒng)等功能[17-18]。近來研究發(fā)現(xiàn)在神經(jīng)退行性疾病和各種腦功能紊亂疾病中有起到防治作用[19-20]，并證實Cur能保護多種細胞免受H2O2誘導的氧化損傷[21-22]。但關(guān)于Cur對神經(jīng)細胞氧化應激損傷的保護作用機制的探討相對較少[23-24]。如前所述，HO-1與nNOS在一些病理狀態(tài)下與氧化應激損傷存在著密切關(guān)聯(lián)，因此，本實驗采用H2O2誘導海馬神經(jīng)元氧化應激的損傷模型，觀察Cur對HO-1、nNOS的影響，探討Cur抗氧化應激損傷的保護作用機制，為Cur的臨床應用提供理論依據(jù)。 目的：探討姜黃素對氧化應激損傷海馬神經(jīng)元的保護作用機制，為姜黃素的臨床應用提供一些理論依據(jù)。方法：分離新生SD大鼠(≤24h)的海馬，采用Neurobasal+2％B27無血清原代培養(yǎng)海馬神經(jīng)元，倒置相差顯微鏡觀察海馬神經(jīng)元生長情況、檢測神經(jīng)元純度，MTT法檢測神經(jīng)元活性，NeuN、NF-200進行神經(jīng)元鑒定。培養(yǎng)至10d，將細胞分為6組：空白對照組、損傷組、姜黃素處理組(2.5μmol/L、5.0μmol/L、10.0μmol/L、20.0μmol/L)。等體積H2O2(1mmol/L)處理海馬神經(jīng)元1h，建立氧化應激損傷模型；用DMEM/F12輕洗2次，姜黃素各處理組分別加入濃度為2.5μmol/L、5.0μmol/L、10.0μmol/L、20.0μmol/L的姜黃素培養(yǎng)液300μl，空白對照組和損傷組分別加入無血清培養(yǎng)基300μl，培養(yǎng)孵育6h后利用倒置相差顯微鏡觀察細胞變化，MTT法測定各組神經(jīng)元活性，乳酸脫氫酶比色法和硫代巴比妥酸法測定LDH活力和MDA濃度，免疫組化和免疫熒光測定海馬神經(jīng)元HO-1、nNOS的表達，RT-PCR測定HO-1、nNOS的mRNA表達。結(jié)果：1.海馬神經(jīng)元接種在培養(yǎng)板上3~4h細胞貼壁，第1d細胞長出突起，第7~8d細胞突觸生長迅速，第9~11d細胞交織成網(wǎng)狀，細胞生長旺盛；20d后細胞軸突縮短、斷裂、胞體無光暈。利用無血清培養(yǎng)基和差速貼壁方法培養(yǎng)的神經(jīng)元純度可達92%。MTT法檢測較其他各時間點的神經(jīng)元活性，9~11d的海馬神經(jīng)元活性較高，P 0.05，利于海馬神經(jīng)元氧化應激損傷模型建立。2.倒置相差顯微鏡觀察海馬神經(jīng)元氧化應激損傷模型(1h)，細胞突觸粘附力降低，部分細胞漂浮，軸突粗糙、斷裂，細胞內(nèi)可見空泡。3.6h后倒置相差顯微鏡下觀察各組細胞，5.0μmol/L組、10.0μmol/L組細胞形態(tài)與1h時比較無明顯變化；2.5μmol/L組、20.0μmol/L組軸突縮短比1h時明顯，但軸突無斷裂、胞體仍有光暈；損傷組細胞軸突縮短、斷裂、胞體光暈減低比1h時更明顯、數(shù)量更多；空白對照組細胞軸突完整、光滑、交織成網(wǎng)狀、胞體光暈強度與1h時無變化。4. MTT檢測5.0μmol/L組和10.0μmol/L組較損傷組神經(jīng)元活力明顯增高，P 0.05；較損傷組，空白對照組神經(jīng)元活力更強，P 0.05；5.0μmol/L組和10.0μmol/L組神經(jīng)元活力與2.5μmol/L組、20.0μmol/L組神經(jīng)元活力比較，差異有統(tǒng)計學意義，P 0.05；2.5μmol/L組、20.0μmol/L組較損傷組神經(jīng)元活力比較，差異無統(tǒng)計學意義，P0.05。5. LDH活力檢測5μmol/L組和10μmol/L組與損傷組比較LDH活力降低，P 0.05；損傷組與空白對照組比較LDH活力增強，P 0.05；5.0μmol/L組和10.0μmol/L組LDH活力與2.5μmol/L組、20.0μmol/L組LDH活力比較，差異有統(tǒng)計學意義，P 0.05；2.5μmol/L組和20.0μmol/L組與空白對照組比較LDH活性降低，P 0.05。6. MDA濃度檢測5.0μmol/L組和10.0μmol/L組與損傷組比較MDA濃度降低，P 0.05；損傷組與空白對照組比較MDA濃度升高，P 0.05；5.0μmol/L組和10.0μmol/L組MDA濃度與2.5μmol/L組、20.0μmol/L組MDA濃度比較，差異有統(tǒng)計學意義，P 0.05；2.5μmol/L組和20.0μmol/L組與空白對照組比較MDA濃度升高，P 0.05。7.免疫組化、免疫熒光、PCR檢測與損傷對照組比較，5.0μmol/L組、10.0μmol/L組HO-1表達升高，nNOS表達降低，P 0.05；損傷組與空白對照組比較HO-1表達下降，nNOS表達升高，P 0.05；5.0μmol/L組和10.0μmol/L組HO-1、nNOS的表達與2.5μmol/L組、20.0μmol/L組比較差異有統(tǒng)計學意義，P 0.05；2.5μmol/L組、20.0μmol/L組與損傷組比較HO-1、nNOS的表達，差異無統(tǒng)計學意義，P0.05。結(jié)論：1.濃度為5μmol/L和10μmol/L的姜黃素可升高氧化應激損傷海馬神經(jīng)元HO-1的表達、降低nNOS的表達，表現(xiàn)出對神經(jīng)元一定程度的保護作用；2.脂質(zhì)過氧化參與了H2O2誘導的海馬神經(jīng)元的氧化應激性損傷，姜黃素處理組5μmol/L和10μmol/L可降低神經(jīng)元的脂質(zhì)過氧化，減少神經(jīng)元LDH的釋放，反映出對神經(jīng)元細胞膜的保護作用。
[Abstract]:Research background
Heme oxygenase (HO) is a speed limiting enzyme in heme catabolism. The CO in human body is mainly produced by the metabolism of heme oxygenase (HO), which produces three types of.HO: inducible (HO-1), composition type (HO-2) and unspecified HO-3[1]. studies, and HO-1 not only plays a role in the physiological state of the body, but also mainly in the physiological state of the body. .HO-1 participates in the oxidative stress response induced by active oxygen, active nitrogen, ischemia, lipopolysaccharide and heme in other abnormal state / stress state. The study shows that the increase of HO-1 expression can reduce the brain tissue morphology and oxidative stress injury in rats with stress injury, and the mechanism may be associated with Nrf2-Keap1 (NF-E2-relatedfact Or2, transcription factor NF-E2 related factor 2) - (Kelch-likeECH associated protein1, Cytoplasmic Protein chaperone) and PI3K/Akt/GSK-3 beta (phosphatidyllinositol-3-kinase/Akt/glycogen syntheses kinase-3 beta, phosphatidyl inositol kinase -3/ silk threonine kinase / glycogen synthetase kinase -3 beta) pathway related to the transcription of the [2-3]. heme They are decomposed into bilirubin, free iron ions and CO. These substances are involved in the removal of free radicals, inhibition of inflammatory factors, up regulation of transcription activator, phosphorylation of tau protein, and inhibition of the ubiquitination of HO-1, [4-7].HO-2 is mainly distributed in the central nervous system and testis, and the CO produced by it is transmitted to nerve signals. It plays an important role, which is closely related to the role of CO in neurotransmitters. HO-2 maintains normal physiological functions of the brain, while HO-1 is closely related to nervous system diseases.
Nitric oxide syntheses (NOS) has three isozymes, including neural nitric oxide synthase (nitric oxide syntheses, nNOS), which mainly exists in the nervous system, and exists in macrophages, hepatocytes and glial cells, the inducible nitric oxide synthase (nitric oxidesyntheses, eNOS) and mainly in the inside. The endothelial nitric oxide synthase (nitricoxide syntheses, iNOS) in the skin cells. In vivo, NOS uses L- arginine and molecular oxygen as a substrate, NADPH coenzyme as an auxiliary factor, and produces nitric oxide (NO) and citrullinate through a series of oxidative reactions. In the nervous system, NO as an important signal molecule, participates in learning and Memory and other important neurophysiological activities also regulate the blood flow of the brain and participate in the immune defense of the nervous system. On the other hand, excessive NO produces cytotoxic oxidizing substances (such as nitrogen radical, peroxy nitrite, etc.), which are closely related to the occurrence and development of diseases such as cerebral ischemia, dementia, Parkinson's disease and so on. Under normal physiological conditions, there are mechanisms in the nervous system that regulate NO production, release, diffusion and inactivation from time and space, and this is mainly realized by regulating activation and deactivation of nNOS. It has been proved that nNOS directly regulates nNOS by combining with CB2 (type2cannabinoidreceptor, cannabinoid receptor 2) and then with antioxidant eggs. White Hsp70 and anti apoptotic protein Bcl-2 jointly complete the CB2 pathway, and delayed neurodegenerative [8]. nitric oxide synthase regulates the free radical of nitric oxide and oxygen free radical balance [9] in the body. It participates in post-traumatic injury and nerve regeneration, ischemia reperfusion injury, neurodegenerative disease, stroke, schizophrenia and other pathophysiological processes. [10-13], its mechanism is associated with lipid peroxidation, protein carbonylation, protein nitroylation and free radical scavenging related [14-15].R OS R and other [16] to explore the central damage model of low arsenic poisoning rats. Not only the reactive oxygen species and lipid peroxidation products can be used as the biological markers of oxidative stress damage, but nNOS can also be used as a result of oxidative stress injury. One of the symbols of physical science.
Curcumin (Cur) is a kind of fat soluble polyphenols isolated from the rhizomes of the ginger family Curcuma longa. It has antioxidative, anti-inflammatory, anti-tumor, anti fibrosis, the protection of cardiovascular system and digestive system and other functions of [17-18]. recently found in the neurodegenerative disease and various brain disorders to prevent the disease. The treatment of [19-20], and confirmed that Cur can protect a variety of cells from H2O2 induced oxidative damage [21-22]., but the mechanism of the protective action of Cur on oxidative stress injury of nerve cells is relatively less [23-24]. as mentioned earlier, HO-1 and nNOS are closely related to oxidative stress damage in some pathological conditions, therefore, H2 in this experiment is used in this experiment. O2 induced oxidative stress damage model of hippocampal neurons, observed the effect of Cur on HO-1 and nNOS, and explored the protective mechanism of Cur antioxidant stress injury, and provided a theoretical basis for the clinical application of Cur.
Objective: To investigate the protective mechanism of curcumin on hippocampal neurons damaged by oxidative stress, and to provide some theoretical basis for the clinical application of curcumin. Methods: the hippocampus of newborn SD rats (< < 24h) was isolated and hippocampal neurons were cultured with Neurobasal+2%B27 serum-free primary culture, and the growth of hippocampal neurons was observed by phase contrast microscope. The neuron purity was detected, the neuron activity was detected by MTT method, and the neuron was identified by NeuN and NF-200. The cells were cultured to 10d, and the cells were divided into 6 groups: blank control group, injury group, curcumin treatment group (2.5 mol/L, 5 mu mol/L, 10 mu mol/L, 20 Mu mol/L). 2 light washing for 2 times, the treatment groups of curcumin were treated with the concentration of 2.5 mu mol/L, 5 mu mol/L, 10 mu mol/L, 20 mol/L curcumin culture medium 300 mu L. The blank control group and the injured group were added to the serum-free culture medium 300 micron. After incubating 6h, the cell changes were observed by the inverted phase contrast microscope, and the activity of neurons in each group was determined by MTT method and lactic acid was removed. LDH activity and MDA concentration were measured by hydrogen enzyme colorimetric assay and thiobarbituric acid method. Immunohistochemistry and immunofluorescence were used to determine the expression of HO-1 and nNOS in hippocampal neurons. RT-PCR was used to determine the mRNA expression of HO-1 and nNOS. Results: 1. the hippocampal neurons were inoculated on the culture board, the 3~4h cells were adhered to the culture board, the 1D cells grew out, the 7~8d cell synapses grew rapidly, the 9~11d cell synapses were fast, the 9~11d cells were 9~11d. The cells were interwoven into a network, and the cells grew exuberant. After 20d, the cell axons were shortened, broken, and the cell body had no halo. The purity of the neurons cultured with the serum-free medium and the differential adherence method could reach the neuron activity of the 92%.MTT method compared with the other time points. The activity of the hippocampal neurons in the 9~11d was higher, and the P 0.05 was beneficial to the oxidative stress in the hippocampus neurons. The damage model established the.2. inverted phase contrast microscope to observe the oxidative stress damage model of hippocampal neurons (1H). The adhesion force of the synapse was reduced, some cells were floating, the axons were rough and fractured. The cells were observed under the inverted phase contrast microscope in the cell. The cell morphology of the group of 5 mu mol /L and the group of 10 mu mol/L had no obvious change. The axon shortening in the group of 2.5 mol/L and 20 mu mol/L was obviously shorter than that of 1H, but the axon was not fractured and the cell body still had halo, and the cell axons were shortened, broken and the cell body halo decreased more obviously than that of 1H, and the number of cell axons in the blank control group was more complete, smooth and interwoven into the network, and the cell body halo intensity was not changed by.4. MTT to detect 5 u Mo when 1H. The neuron vigor of the group l/L and the 10 mol/L group was significantly higher than that in the injured group, P 0.05. Compared with the injury group, the neuron vigor was stronger and the P 0.05. The neuron vigor of the 5 mu mol/L group and the 10 micron mol/L group was compared with the 2.5 mu mol/L group and the 20 u mol/L group. The difference was statistically significant, P 0.05, 2.5 mu mol/L group and 20 u mol/L group were more damaged. There was no significant difference in the activity of neuron in the injured group. The activity of P0.05.5. LDH was compared with the group of 5 mu mol/L and the 10 mol/L group, the activity of LDH was decreased and P 0.05 was compared with that of the injury group. The activity of LDH was enhanced and P 0.05 was compared with the blank control group, and the LDH vigor of the 5 mu group and the 10 mu mol/L group was compared with the 2.5 mu mol/L group, and the difference between the 20 mu group and the group was different. Statistical significance, P 0.05; 2.5 mu mol/L group and 20 mu mol/L group compared with blank control group, LDH activity decreased, P 0.05.6. MDA concentration was detected in 5 mu mol/L group and 10 micron group, and MDA concentration decreased and P 0.05 compared with injury group. The concentration of MDA in the injured group was higher than that in the blank control group, and 0.05, 5 mu and 10 micron groups were compared with 2.5 mu. In group ol/L, the concentration of MDA in group 20 mol/L was statistically significant, the difference was statistically significant, P 0.05; 2.5 mu mol/L group and 20 mol/L group were compared with the blank control group, MDA concentration increased, P 0.05.7. immunization, immunofluorescence, PCR detection compared with the damage control group, 5 mu mol/L group, 10 mu mol/L group increased expression, 0.05; injury group and space In the white control group, the expression of HO-1 decreased, the expression of nNOS increased, the expression of P 0.05, the 5 mu mol/L group and the 10 mu mol/L group HO-1, the nNOS expression was statistically significant compared with the 2.5 mu mol/L group, and the 20 micron mol/L group. The 20 micron mol/L group was compared with the injury group, and the difference was not statistically significant. 1. concentration was 5 mu. Mol/L and 10 mol/L curcumin can increase the expression of HO-1 in hippocampal neurons of oxidative stress, reduce the expression of nNOS, and show a certain protective effect on neurons; 2. lipid peroxidation is involved in oxidative stress injury induced by H2O2 induced hippocampal neurons. The lipid peroxidation in the group of curcumin can reduce the lipid of neurons. Mass peroxidation can reduce the release of LDH in neurons and reflect the protective effect on neuronal cell membrane.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741

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相關(guān)期刊論文 前1條

1 陳秀,李作孝,張琳,佟琳,陳忠倫;姜黃素對腦出血大鼠血腫周圍腦組織氧化損害保護作用的研究[J];腦與神經(jīng)疾病雜志;2004年05期

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