南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)Journal of Nantong University (Medical Sciences) 2013: 33(4)283氯胺酮與乙醇對(duì)小鼠海馬PKA-CREB信號(hào)通路的影響楊美玉13,丁飛2,蔣小崗3,顧振綸3,卞士中1(蘇州大學(xué)醫(yī)學(xué)部法醫(yī)學(xué)系,蘇州215123;2上海市公安局寶山分局刑事科學(xué)研究所;3蘇州中藥研究所)摘要]目的:研究氯胺酮與乙醇對(duì)小鼠海馬ⅣKA-CREB信號(hào)通路的影響,探討氯胺酮與乙醇致學(xué)習(xí)記憶障礙的分子學(xué)機(jī)制。方法:40只ICR小鼠隨機(jī)分為4組:對(duì)照組、氯胺酮組、乙醇組、氯胺酮乙醇合用組,每組10只。氯胺酮和乙醇分別采取腹腔注射和灌胃的給藥方式,1次,連續(xù)14d。通過(guò)RT-PCR檢測(cè)海馬中c-AMP反應(yīng)單元結(jié)合蛋白(c- AMP response element binding protein,CREB)、蛋白激酶A( protein kinase A,PA)及腺苷酸環(huán)化酶( adenylate cyclase,AC)mRNA的表達(dá); Western blot檢測(cè) pCREB/CREB的蛋白表達(dá)。結(jié)果:RT-PCR檢測(cè)結(jié)果:氯胺酮乙醇合用組可顯著降低海馬區(qū)CREB、PKA及 AC mrNA的表達(dá)(P<0.05), Western Blot檢測(cè)顯示 PCREB/CREB的蛋白表達(dá)顯著降低(P<0.05)。結(jié)論:氯胺酮與乙醇可協(xié)同抑制小鼠的學(xué)習(xí)記憶行為,其機(jī)制可能與大腦海馬區(qū)CREB信號(hào)通路相關(guān)[關(guān)鍵詞]氯胺酮;乙醇;學(xué)習(xí)記憶;環(huán)磷酸腺苷反應(yīng)結(jié)合蛋白;小鼠[中圖分類(lèi)號(hào)]R971[文獻(xiàn)標(biāo)志碼]A[文章編號(hào)]1674-7887(2013)04-0283-05Effects of ketamine on ethanol-induced learning and memory impairment in miceYANG Meiyu., DING Fei JIANG Xiaogang, GU Zhenlun, BIA N Shizhong(Department of Forensic MedicineMedical College oflow University, Institute of Forensic Sciences of Soochow University, Suzhou 215123; Institute ofCriminal Sciences, Baoshan Branch of Shanghai Public Security Bureau; Suzhou Institute of Chinese Materia Medica[Abstract] Objective: To study the effects of ketamine and ethanol on PKA-CREB pathway of hippocampus -dependentlearning and memory in mice and its possible mechanism. Methods: A total of 40 male mice were divided into 4 groups: anormal control group, a ketamine group, an ethanol group and an ethanol plus ketamine group. Ketamine and ethanol wererespectively taken by intraperitoneal injection and oral administration, once per day, for 14 d. The expressions of CreBPKA and AC gene in hippocampus were detected by RT-PCR. The expressions of pCrEB/CREB were detected by WesterBlot. Results: After the combined treatment with ketamin and ethanol. creb. pKa and ac mrnA were down regulated byRT-PCR(P<0.05), and pCREB/CREB were down regulated by Western Blot(P<0.05 ). Conclusion: The data indicatethat combination of ketamine and ethanol is synergic in the inhibition of learning and memory in mice, its mechanism maybe related to the CReB signal transduction pathway[Key words] ketamine; ethanol; learning and memory C-AMP-response element binding protein; mouse氯胺酮是N-甲基-D-天(門(mén))冬氨酸(N- methyl-入細(xì)胞核磷酸化CREB,然后激活I(lǐng)Gs(C-fos)等,其D- aspartate,NMDA受體的非特異性阻斷劑,其作激活產(chǎn)物進(jìn)一步編碼LTP所需要的蛋白質(zhì)。用機(jī)制復(fù)雜。日前研究認(rèn)為,麻醉劑量的氯胺酮本研究以小鼠為實(shí)驗(yàn)對(duì)象,觀察氯胺酮與酒精可通過(guò)阻斷NMDA受體抑制學(xué)習(xí)記憶,該受體對(duì)神聯(lián)合給藥對(duì)小鼠學(xué)習(xí)記憶的影響。采用RT-PCR技經(jīng)元突觸長(zhǎng)時(shí)程增強(qiáng)(ong- -term potentiation,TP)的術(shù)檢測(cè)小鼠海馬中CREB、PKA及腺甘酸環(huán)化酶產(chǎn)生、維持和學(xué)習(xí)記憶功能至關(guān)重要。adenylate cyclase, AC)mRNA的表達(dá); Western blot令A(yù)環(huán)磷腺 cyclin adenosine monophosphate, C-AMP))檢測(cè) pCREB/CREB的蛋白表達(dá)情況,以探討氯胺酮應(yīng)元件結(jié)合蛋白(e- AMP response element bindi與乙醇對(duì)小鼠學(xué)習(xí)記憶行為學(xué)影響的分子機(jī)制protein,CREB)是一種依賴(lài)c-AMP的轉(zhuǎn)錄因子,可將細(xì)胞內(nèi)的c-AMP的量的變化轉(zhuǎn)錄為基因表達(dá)的1材料與方法形式。CREB在記憶的形成過(guò)程中發(fā)揮了及其重要1.1實(shí)驗(yàn)動(dòng)物ICR小鼠40只,雄性,體質(zhì)量25~的作用,其過(guò)程可能是:神經(jīng)遞質(zhì)等作用神經(jīng)元細(xì)30g蘇州大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。實(shí)驗(yàn)前小鼠適應(yīng)胞,導(dǎo)致胞質(zhì)內(nèi)c-AMP濃度升高,使蛋白激酶A性飼養(yǎng)3d,維中國(guó)煤化仝實(shí)驗(yàn)過(guò)程中protein kinase A,PKA)激活,PKA的催化亞單位進(jìn)小鼠自由飲水CNMHG*[作者簡(jiǎn)介]楊美玉,女,漢族,生于1984年3月,山東省濟(jì)寧市人,碩土在讀,研究方向:法醫(yī)毒理病理學(xué)*[通信作者]卞士中,電話:0512-67580893,E- mail hanshizhong@ suda. edu. en284南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)2013:33(4)1.2主要試劑及儀器鹽酸氯胺酮注射液(國(guó)藥準(zhǔn)表2內(nèi)參和目的基因擴(kuò)增條件字H32022820,批號(hào):KH071004,2ml0.1g)購(gòu)自江基因擴(kuò)增條件蘇恒瑞醫(yī)藥公司;56%v紅星二鍋頭酒購(gòu)自北京紅 GAPDH94℃4min94℃30s-55℃3072℃星股份有限公司;PCR擴(kuò)增試劑盒(批號(hào):DRRO0Almin→72℃10min+4℃抗體(抗10購(gòu)p兔抗小鼠CREB米W多購(gòu)自 TaKara公司;RNA逆轉(zhuǎn)錄引物:由上海生物工94℃5min+94℃1min→57℃30s72℃程有限公司合成;兔抗鼠 phospho-CREB-1(Ser13345s-+72℃10min+4℃94℃4min+94℃30s→57℃30s-72℃(30 cycleslmin→+72℃10min+4℃942min→94℃30s→55℃45s72℃actin(1:1000購(gòu)自 Santa cruze;所用抗鼠和抗兔熒45s-+72℃7min-+4℃光二抗均為 Odyssey公司產(chǎn)品5 Western blot1.3模型制備與給藥40只小鼠隨機(jī)分成4組,分1.5.蛋白樣品制備取液氮內(nèi)凍存的小鼠海馬組別為正常對(duì)照組,氯胺酮組,乙醇組,聯(lián)合給藥組,每織約30mg,加入組織裂解液05mL,超聲勻漿后于組10只。給藥方式:對(duì)照組以生理鹽水腹腔注射+生4℃,12000r/min離心10min,吸取上清,分裝,另理鹽水灌胃,乙醇組以生理鹽水腹腔注射+乙醇灌外留少許待測(cè)蛋白溶液(于-40℃冷藏。胃,氯胺酮組以氯胺酮腹腔注射+生理鹽水灌胃,聯(lián)1.5:2BCA試劑盒檢測(cè)蛋白濃度(1)蛋白標(biāo)準(zhǔn)曲合給藥組以氯胺酮腹腔注射←乙醇灌胃。生理鹽水、線繪制取標(biāo)準(zhǔn)蛋白液,用PBS梯度稀釋成4、8、12、氯胺酮溶液(50mgkg)腹腔注射1次ld,連續(xù)14d;生16、20、24μgμL蛋白溶液,每組40μ。取BCA試?yán)睇}水、乙醇紅星二鍋頭稀釋至20%v)灌胃1次,劑盒A液和B液按50:1配成工作液。稀釋后的標(biāo)連續(xù)14d。各組小鼠每日9:00給藥。準(zhǔn)蛋白液與BCA工作液按1:8混合均勻后,37℃水1.4PCR檢測(cè)浴30min,于酶標(biāo)儀上測(cè)定OD值,每組設(shè)3復(fù)孔14.1總RNA提取取液氮內(nèi)凍存的小鼠海馬組由OD值,繪制蛋白標(biāo)準(zhǔn)曲線;(2)待測(cè)蛋白濃度的測(cè)織約30mg,加人 Trizol試劑0.5m,按說(shuō)明書(shū)進(jìn)行定取待測(cè)蛋白液每組10μL。將待測(cè)蛋白液與BCARNA提取,所用RNA的D3D2均在1.8-2.0。工作液按1:8混勻后,37℃水浴30min,于酶標(biāo)儀上1.4.2模板cDNA合成提取的總RNA逆轉(zhuǎn)錄合測(cè)其OD值,每組設(shè)3復(fù)孔。根據(jù)OD值,于蛋白標(biāo)成cDNA。準(zhǔn)曲線上計(jì)算相關(guān)蛋白濃度。根據(jù)蛋白濃度測(cè)定值,1.4.3 RT-PCR調(diào)整樣本上樣量,使各組蛋白量為60μg,按4:1與14.3.1內(nèi)參和目的基因的引物序列及產(chǎn)物長(zhǎng)度5× Loading buffer混勻,水浴100℃變性5min后見(jiàn)表1。冰浴備用。14.32內(nèi)參和目的基因擴(kuò)增條件均為25μL1.5.3SDS-PAGE電泳先將實(shí)驗(yàn)用玻片洗凈,再反應(yīng)體系,應(yīng)用 GAPDH作為參照。內(nèi)參和目的基用無(wú)水乙醇脫脂,晾干后組裝制膠板。配12%的分離因擴(kuò)增條件見(jiàn)表2。PCR產(chǎn)物用1.5%瓊脂糖凝膠膠,注入制膠板中,加適量無(wú)水乙醇封閉。待膠凝固進(jìn)行電泳,結(jié)束后于全自動(dòng)凝膠成像分析系統(tǒng)進(jìn)倒去乙醇,用蒸餾水清洗3次。配制濃縮膠,兩端交行灰度掃描。以 GAPDH擴(kuò)增產(chǎn)物校正作光密度相替加入制膠板,插入梳子后放置約1h待膠凝固后對(duì)分析。使用。安裝電泳裝置,上樣品,進(jìn)行電泳,濃縮膠時(shí)設(shè)表1內(nèi)參和目的基因的引物序列及產(chǎn)物長(zhǎng)度置電壓90V,染料條帶至分離膠時(shí)電壓設(shè)置至基因引物110V,染料接近分離膠底部,停止電泳。F5'-CCGGAACATGGACCTCTACTAC-31.54轉(zhuǎn)膜將丙烯酰胺凝膠從玻璃板上剝離,置R5'-ATAGGTGGGAGGAGATGGACTT-3入轉(zhuǎn)移緩沖液中,將海綿、濾紙、凝膠、NC膜、濾紙、F5'-GGAGAGCGTGAAAGAGTTCCTA-3海綿按順序疊加,用玻璃棒小心趕走氣泡,放置于轉(zhuǎn)(339 bp) R5 -CCAGCTACATACTCCATGACCA移電泳槽中恒流400mA,轉(zhuǎn)移120min。轉(zhuǎn)膜后CREBF5 -AGTATGCACAGACCACTGATGG-3用1×TBST洗中國(guó)煤化工(392 bp) R5 -TCCACCGTAACAGGAGAGAACT-3155封閉10min,然后GAPDH5 -GCCATCAACGACCCCTTCATT-3將膜浸于5%胱CNMHG5'-CGCCTGCTTCACCACCTTCTT-31.5.6抗體孵育封閉結(jié)束后再用1 XTBST洗膜3楊美玉,等氯胺酮與乙醇對(duì)小鼠海馬PKA-CREB信號(hào)通路的影響次,每次10min。將NC膜放入小塑料袋內(nèi),加人5%的降低用統(tǒng)計(jì)學(xué)意義(P<0.05,圖2)。提示:氯胺酮脫脂牛奶按比例稀釋后的一抗,2mL膜。去除袋內(nèi)飲酒、氯胺酮+飲酒聯(lián)合給藥均可下調(diào) pCREB氣泡,用封膜機(jī)封口,置于搖床上室溫孵育3h過(guò)CREB蛋白表達(dá),且聯(lián)合用藥組 PCREB/CREB降低夜。取膜,用1×TBST洗滌3次,每次10min后,用更明顯。5%脫脂牛奶按1:10000的比例稀釋的二抗封閉,置對(duì)照組乙醇氯胺酮氯胺酮+乙醇于搖床上,室溫避光孵育1h。取膜,用1×TBST在暗REB-42 k盒中洗滌3次,每次10min。CReB-42k1.5.7顯影近紅外雙色激光成像系統(tǒng)顯影。42 ku1.6圖像處理及數(shù)據(jù)分析RT-PCR, Western blot圖像掃描后均采用 SigmaScan pro5軟件系統(tǒng)分析,圖2氯胺酮與乙醇對(duì)小鼠海馬CREB, PCREB蛋白表達(dá)的實(shí)驗(yàn)數(shù)據(jù)以x±s表示。顯著性檢驗(yàn)采用 One way影響ANOVA,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。3討論2結(jié)果CREB是堿性氨基酸亮氨酸(Leu拉鏈轉(zhuǎn)錄因子2.1RT-PCR檢測(cè)結(jié)果在連續(xù)給藥14d,小鼠海家族,受細(xì)胞內(nèi)多條信號(hào)通路的調(diào)節(jié),與生長(zhǎng)抑素馬組織RT-PCR檢測(cè)結(jié)果分別以PKA、AC、CREB基因的cAMP反應(yīng)元件( cAMP response element,CRE)與內(nèi)參基因 GAPDH的密度比值表示組織中PKA、有高度親和力。CREB家族在結(jié)構(gòu)上具有DNA結(jié)AC、 CREB mRNA的相對(duì)表達(dá)強(qiáng)度。實(shí)驗(yàn)結(jié)果表明,合結(jié)構(gòu)域和轉(zhuǎn)錄激活結(jié)構(gòu)域,因而具有DNA結(jié)合活與正常對(duì)照組相比,飲酒、氯胺酮、氯胺酮+飲酒聯(lián)合性和轉(zhuǎn)錄調(diào)節(jié)活性。但只有當(dāng)Ser133被PKA、蛋白給藥可誘導(dǎo)小鼠海馬組織中的PKA、AC、CREB激酶 C(protein kinase C,PKC或鈣/鈣調(diào)蛋白激酶(Ca2mRNA表達(dá)下調(diào)均P<0.05)。與單用藥組相比,聯(lián)合 calmodulin- dependent protein kinase,Ca2CaMK)等磷給藥組小鼠海馬組織RKA、AC、 CREB mrNA表達(dá)酸化后才能啟動(dòng)轉(zhuǎn)錄,磷酸化的CREB形成二聚體顯著下降(均P0.05圖1)。提示:飲酒、氯胺酮、氯后活化,并與靶基因CRE序列結(jié)合,調(diào)節(jié)其轉(zhuǎn)錄,發(fā)胺酮+飲酒聯(lián)合給藥可使小鼠海馬組織中PKA、AC、揮多種生物學(xué)效應(yīng)CREB及其相應(yīng)的基因表達(dá)下調(diào),其中氯胺酮+飲酒在中樞神經(jīng)系統(tǒng)中,CREB調(diào)節(jié)神經(jīng)細(xì)胞的生聯(lián)合給藥組作用更明顯長(zhǎng)發(fā)育及營(yíng)養(yǎng),參與神經(jīng)細(xì)胞突觸可塑性和長(zhǎng)時(shí)程Marker對(duì)照組乙醇氯胺酮氯胺酮+乙醇記憶的形成⑦,是長(zhǎng)時(shí)程記憶和LTP所必需的基因400bpGAPDH開(kāi)關(guān)。CREB通過(guò)調(diào)控目的基因的表達(dá),影響晚時(shí)500bp相的LTP和長(zhǎng)時(shí)程記憶。實(shí)驗(yàn)證實(shí)CREB是學(xué)習(xí)記憶中一個(gè)關(guān)鍵因子,400bp300bp參與海馬的空間記憶調(diào)節(jié)。研究認(rèn)為迷宮實(shí)驗(yàn)訓(xùn)練可使大鼠海馬和紋狀體內(nèi) PCREB免疫活性顯著500bp·升高。海馬CREB功能降低,可導(dǎo)致空間記憶功能的400bD減退。譚蕾等研究結(jié)果表明氯胺酮通過(guò)下調(diào)大鼠海馬神經(jīng)元 pCREB水平,誘導(dǎo)新生大鼠海馬神經(jīng)元凋亡,突觸形成受損。 S.C. Pandey等四研究發(fā)現(xiàn)大鼠過(guò)度飲酒,可導(dǎo)致杏仁核CRE-DNA結(jié)合力下降,海圖1氯胺酮與乙醇對(duì)小鼠海馬PKA、AC、CREB基因表達(dá)的影響馬內(nèi)總CREB及 pCREB表達(dá)下調(diào),進(jìn)而影響大鼠的2.2 Western blot檢測(cè)結(jié)果CREB的磷酸化水平學(xué)習(xí)記憶功能。與學(xué)習(xí)記憶密切有關(guān)。連續(xù)給予氯胺酮、飲酒、聯(lián)合氯胺酮為苯環(huán)已哌啶(N-1- phenycyclohexy-給藥14d后,與正常對(duì)照組相比,各用藥組小鼠海 piperidine,PCP且NMnA嚴(yán)體的非特異馬組織 PCREB、CREB蛋白表達(dá)量均降低(P<05,性阻斷劑。氮中國(guó)煤化工通過(guò)阻斷各且磷酸化水平 DCREB/CREB I的降低有統(tǒng)計(jì)學(xué)意義刺激產(chǎn)生的長(zhǎng)CNMHG記憶過(guò)程。有P<005)。與單用藥組相比,聯(lián)合用藥組 PCREB/CREB報(bào)道稱(chēng)氯胺酮可致其濫用者記憶的長(zhǎng)久損害作用286南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)2013:33(4)經(jīng)多次氯胺酮麻醉的大鼠其空間學(xué)習(xí)記憶能力明顯小鼠學(xué)習(xí)記憶的影響[J中國(guó)藥理學(xué)通報(bào),199915(3)下降,且海馬神經(jīng)元超微結(jié)構(gòu)也有異常改變,損害程度與使用氯胺酮的劑量水平有關(guān)叫。本實(shí)驗(yàn)室研究結(jié)5 I Molkentin JI, Dorn GW. Cytoplasmic signaling pathways果表明,氯胺酮多次給藥可致小鼠空間學(xué)習(xí)記憶能that regulate cardiac hypertrophy [J|. Annu Rev Physiol力障礙。2001,63(63:391-426Wu GY, Deisseroth K, Tsien Rw. Activity -dependent在海馬內(nèi),乙醇通過(guò)抑制NMDA受體亞型CREB Phosphorylation: con vergence of a fast, sensitiveNR2B的活性來(lái)抑制NMDA受體嗎。早期研究表明calmodulin kinase pathway and a slow, less sensitive mito乙醇在較低濃度時(shí)就可對(duì)NMDA受體的一個(gè)亞型gen-activated protein kinase pathway[J. Proe Natl Acad起作用,以某種方式改變?cè)撌荏w亞型之間的相互配Scius a,2001,98(5):2808-2813合,從而使谷氨酸利用受體進(jìn)行的信號(hào)傳遞受到破7] Setlow b, Roozendaal b, McGaugh JL. Involvement of a壞,進(jìn)而影響包括LTP在內(nèi)的分子信號(hào)傳導(dǎo)通路受basolateral amygdala complex-nucleus accumthway阻,導(dǎo)致學(xué)習(xí)記憶功能障礙。體外研究證明,乙醇可明顯降低CREB的活性dation(J. Eur J Neurosci, 2000, 12(1): 367-375本研究分子生物學(xué)實(shí)驗(yàn)研究結(jié)果表明:氯胺酮[8 Puzzo D, Vitolo O, Trinchese F, et al. Amyloid-beta pep乙醇、氯胺酮聯(lián)合乙醇作用小鼠14d后,小鼠海馬tide inhibits activation of the nitric oxide/cGMP/e AMP-re-組織AC、PKA、 CREB mRNA的表達(dá)減少,且兩兩比onsive element-binding protein pathway during較顯示聯(lián)合用藥組與單用藥差異均有統(tǒng)計(jì)學(xué)意義,pocampal synaptic plasticity[J]. 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J Neural Transm, 2005, 112(8)南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)Journal of Nantong University (Medical Sciences) 2013: 33(4)Wnt5a在嚴(yán)重燙傷大鼠早期腸黏膜組織中的表達(dá)及胰島素的調(diào)控作用祁俊”,胡克蘇,張?jiān)?張逸(南通大學(xué)附屬醫(yī)院燒傷整形科,南通226001)摘要]目的:研究在嚴(yán)重燙傷大鼠腸黏膜組織中Wnt5a的動(dòng)態(tài)表達(dá)變化及其意義。方法:將48只大鼠背部浸入恒溫水浴盆⑨2℃中20s達(dá)Ⅲ度燙傷,制作燙傷創(chuàng)面,采用隨機(jī)數(shù)字法將大鼠分為對(duì)照組、單純燙傷組燙傷組)、燙傷后胰島素治療組(治療組)。傷后取腸組織觀察大體變化,取腸道黏膜H染色后,行組織形態(tài)學(xué)觀察,分別于燙傷后6、1224、48h觀察,取腸黏膜組織以免疫組織化學(xué)的方法,檢測(cè)Wnt5a的表達(dá)情況。結(jié)果:在燙傷組和治疔組Wnt5a表達(dá)均有顯著表達(dá),對(duì)照組有少量表達(dá)(P<005),傷后6-12hWn5a的表達(dá)持續(xù)升高,燙傷組12-24h開(kāi)始表達(dá)逐漸減少,24h后表達(dá)又重新增多,各組在48h表達(dá)達(dá)到最高峰;與燙傷組相比,治療組Wnt5a的表達(dá)水平明顯降低(P<0.05),病理形態(tài)學(xué)變化顯示燙傷早期大鼠腸黏膜組織損傷明顯,治療組大鼠腸黏膜組織損傷較燙傷組明顯減輕。結(jié)論:嚴(yán)重燙傷早期SD大鼠腸黏膜組織損傷眀顯,wnt5a可能參與了燒傷膿毒癥的發(fā)生發(fā)展過(guò)程。胰島素能減少其表達(dá),可能是其保護(hù)腸黏膜機(jī)制的因素之關(guān)鍵詞]燒傷;腸黏膜;Wn5a;大鼠「中圖分類(lèi)號(hào)]R644文獻(xiàn)標(biāo)志碼]A文章編號(hào)]1674-7887(2013)04-0287-04Expression of Wnt5a in intestinal mucosa of rats during the early stage after severe burninjury and the effect of the insulinQI Jun", HU Kesu, ZHANG Yun, ZHANG Yi(Department of Plastic Surgery, the Affiliated Hospital of Nantong University, Nantong 226001)[Abstract] Objective: To investigate the changes of the expressions of Wnt5a in intestinal mucosa of rats during the earlystage after severe burn Injury and its sigficance. Methods: 48 healthy SD rats were set upscalded models, which wererandomly divided into control group, scald group and scald +insulin group(treatment group). The intestinal mucosa tissuesample separately after 6, 12, 24, 48 hours were posted scald injury. In the treatment group, subcutaneous injection of 3 U/kginsulin after scald injury. The expression of Wnt5a in the intestinal mucosa was detected with immunohistochemistry methodand analyzed by a micro-image analysis system, and at the same time we observed the pathological changes of the intestinalmucosa tissue of each groups. Results: In the scalded group, the expression of Wnt5a was higher than that in control group atthe same time (P<0.05),6-12 hours post scald injury the expression was increased. Following the expression of Wnt5adecreased obviously 24 hours post scald injury, 48 hours post scald injury the expression was increased. The expression ofWnt5a in the treatment group was significantly smaller than that in scalded group at most time points at the same time(P<0.05). Conclusions: In the early phase of severe burns injury, the intestinal mucosa tissue injury is very severe. Wnt5amay take part in the procession of burn sepsis. Insulin can deduce its expression, which may be one of factors protecting thetestinal mucosa tissue[ Key words] burn; intestinal mucosa Wnt5a; rat臨床很早就發(fā)現(xiàn)嚴(yán)重的燒傷可導(dǎo)致胃、十二指量的細(xì)菌和內(nèi)毒素,主要是依賴(lài)于其腸道屏障的功腸潰瘍,甚至岀現(xiàn)消化道大出血或穿孔等并發(fā)癥。能。對(duì)其屏障的功能行深人地研究,有可能使燒傷的其影響因素有多種,目前尚未完全明了,胃腸黏膜缺救治水平再上1個(gè)臺(tái)階。Wnt5a為分泌性信號(hào)轉(zhuǎn)導(dǎo)血和胃液氫離子反滲是主要的發(fā)病原因。腸道是人體分子Wnt家族成員。近來(lái),不少研究資料顯示內(nèi)最大的¨內(nèi)毒素庫(kù)”。正常的腸道之所以能抵抗大wnt5a在單核巨噬細(xì)胞的激活中發(fā)揮著重要作用。*[基金項(xiàng)目]南通大學(xué)自然科學(xué)類(lèi)科研課題(11Z017*[作者簡(jiǎn)介]祁俊,男,漢族,生于1980年8月,江蘇省南通市人,碩士,主治醫(yī)師,研究方向:燒傷整形等**[通信作者]張逸,電話:0513-81168901,E- mail: qijunsky@126cmMH中國(guó)煤化工1005-1013.CNMHG6[17] Kumar D, Deb L, Chakraborty J, et al. A polymorphism of桐日期]2013-03-10the CREB binding protein(CREBBP) gene is a risk factor