南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版Journal of Nantong University(Medical Sciences) 2013: 33(4283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”(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。氯胺酮和乙醇分別釆取腹腔注射和灌胃的給藥方式,1次/d,連續(xù)14d。通過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,FKA)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 JIA NG Xiaogang, GU Zhenlun, BlA N Shizhong (Department of Forensic MedicineMedical College of Soochow 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 WesternBlot. Results: After the combined treatment with ketamin and ethanol. CREB. PKA and AC mRNA were down regulated byRT-PCR(P<0. 05), and p CREB/CREB were down regulated by Westem 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)冬氨酸(N- methy-人細(xì)胞核磷酸化CREB,然后激活TEGs(C-os)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。目前研究認(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環(huán)磷腺苷 (cyclin adenosine monophosphate,c-AMP)檢測(cè) pCREB/CREB的蛋白表達(dá)情況,以探討氯胺酮反應(yīng)元件結(jié)合蛋白(c- AMP response element binding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,維持室溫(22±1)℃,在整個(gè)實(shí)驗(yàn)過程中protein kinase A,PKA)激活,PKA的催化亞單位進(jìn)小鼠自由飲水和進(jìn)食。*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. bianshizhongt@ suda. edu. en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):KH071004,2ml:0.1g)購自江基因擴(kuò)增條件蘇恒瑞醫(yī)藥公司;56%v/紅星二鍋頭酒購自北京紅 GAPDH94℃4min94℃30s-55℃30s72℃星股份有限公司;PCR擴(kuò)增試劑盒(批號(hào):DR0A)63lmin+72℃10min+4℃購自 Takara公司;RNA逆轉(zhuǎn)錄引物:由上海生物工℃5min→94℃1min→+57℃30s-72℃程有限公司合成;兔抗鼠 phospho-CREB-1(Ser13345s72℃10min*4℃抗體(一抗)(1:100)購自 slabs;抗小鼠CREB抗體94℃4min→→94℃30s57℃30s+72℃30 cycles)lmin+72℃10min-+4℃(一抗)(1:100購自武漢博士德生物技術(shù)有限公司CREB94℃2min+94℃30s→55℃45s72℃actin(1:1000)購自 Santa cruze;所用抗鼠和抗兔熒(30 cyeles)45s+72℃7min-+4℃光二抗均為 Odyssey公司產(chǎn)品1.5 Western blot1.3模型制備與給藥40只小鼠隨機(jī)分成4組,分1.5.1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05m,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℃,12000 r/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l,連續(xù)14d;生16、20、24μg/μL蛋白溶液,每組40μL。取BCA試?yán)睇}水、乙醇紅星二鍋頭稀釋至20%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。1.4.1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Tiol試劑0.5mL,按說明書進(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D/D2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× padding Buffer混勻,水浴100℃變性5min后,見表1。冰浴備用4.3.2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)増產(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(284 bp) R5'-ATAGGTGGGAGGAGATGGACTT-3入轉(zhuǎn)移緩沖液中,將海綿、濾紙、凝膠、NC膜、濾紙F(tuán)5 '-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-3移電泳槽中恒流400mA,轉(zhuǎn)移120mi。轉(zhuǎn)膜后,CREBF5=AGTATGCACAGACCACTGATGG-3用1×TBST洗膜。(392 bp) R5'-TCCACCGTAACAGGAGAGAACT-31.5.5封閉1×TBST洗膜3次,每次10min,然后GAPDH F5 -GCCATCAACGACCCCTTCATT-3將膜浸于5%脫脂牛奶中封閉1h(413 bp) R5'-CGCCTGCTTCACCACCTTCTT-31.5.6抗體孵育封閉結(jié)束后再用1×TBST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285次,每次l0mins將NC膜放入小塑料袋內(nèi),加入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_抗,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pCREB-盒中洗滌3次,每次10min。15.7顯影近紅外雙色激光成像系統(tǒng)顯影CREB-42k1.6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.lRT-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RKA、AC、CREB激酶 C(protein kinase C,ⅨKC或鈣/鈣調(diào)蛋白激酶Ca27mRNA表達(dá)下調(diào)均P0.05。與單用藥組相比,聯(lián)合 calmodulin- dependent protein kinase, Ca+/CaMK等磷給藥組小鼠海馬組織pKA、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P<0.05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,是長(zhǎng)時(shí)程記憶和LTP所必需的基因開關(guān)閃。CREB通過調(diào)控目的基因的表達(dá),影響晚時(shí)(413bp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)鍵因子參與海馬的空間記憶調(diào)節(jié)。研究認(rèn)為迷宮實(shí)驗(yàn)(284bp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400bp減退。譚蕾等研究結(jié)果表明氯胺酮通過下調(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等研究發(fā)現(xiàn)大鼠(392bp)圖1氯胺酮與乙醇對(duì)小鼠海馬PKA、AC、CREB基因表達(dá)的過度飲酒,可導(dǎo)致杏仁核CRE-DNA結(jié)合力下降,海影響馬內(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)己哌啶(N-1- phenyeyc lohexy-給藥14d后,與正常對(duì)照組相比,各用藥組小鼠海 piperidine,PCP)的衍生物,是NMDA受體的非特異馬組織 PCREB、CREB蛋白表達(dá)量均降低(P<0.05),性阻斷劑。氯胺酮拮抗NMDA受體,可通過阻斷各且磷酸化水平 pCREB/CREB的降低有統(tǒng)計(jì)學(xué)意義刺激產(chǎn)生的長(zhǎng)時(shí)程增強(qiáng)效應(yīng),干擾學(xué)習(xí)記憶過程。有P005與單用藥組相比聯(lián)合用藥組 PCREB/CREB報(bào)道稱氯胺酮可致其濫用者記憶的長(zhǎng)久損害作用。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[中國(guó)藥理學(xué)通報(bào),199,15(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90-93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 Molkentin JI, Dorn gW. Cytoplasmic signaling pathways在海馬內(nèi),乙醇通過抑制NMDA受體亞型2001,636330小 hypertrophy. Annu rey physiol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力障礙。Wu GY, Deisseroth K, Tsien RW. Activity -dependentNR2B的活性來抑制NMDA受體。早期研究表明CREB Phospheof a fast. sensitivecalmodulin kinase pathway and a slow, less sensitive mite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Seius a.2001,985)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 JI. 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 accumbens pathway阻,導(dǎo)致學(xué)習(xí)記憶功能障礙。體外研究證明,乙醇in glucocorticoid-induced modulation of memory consoli可明顯降低CREB的活性。dation. Eur J Neurosci, 2000, 12(1): 367-375本研究分子生物學(xué)實(shí)驗(yàn)硏究結(jié)果表明:氯胺酮、18] Puzo d.iolo. Trinchese h,etal. Amyloid- -beta pep乙醇、氯胺酮聯(lián)合乙醇作用小鼠14d后,小鼠海馬ide inhibits activation of the nitric oxidelegMPicAMP-re組織AC、PKA、 CREB MRNA的表達(dá)減少,且兩兩比onsive element-binding protein pathwaypocampal synaptic plasticity [J). J Neurosci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6887-6897提示氯胺酮和乙醇可能在基因轉(zhuǎn)錄水平上對(duì)CREBLiu ry fioravante d. shah S. et al. cAMP信號(hào)傳導(dǎo)通路協(xié)同抑制。 Western blot檢測(cè)結(jié)果表ment-binding protein 1 feedback loop is necessary for明:用藥14d后,小鼠海馬組織中, pCREB/CREB蛋onsolidation of long-term synaptic facilitation in aplysiaJI白表達(dá)也有所下降,提示氯胺酮和乙醇在蛋白質(zhì)水J Neurosci.,2008,28(8):1970-1976平上影響CREB信號(hào)傳導(dǎo)通路。與本實(shí)驗(yàn)室行為學(xué)10 Colombo PJ, Brightwell JJ. Countryman RA. Cognitive str結(jié)果一致,提示氯胺酮、乙醇可加強(qiáng)抑制小鼠海馬組ategy -specific increases in phosphorylated cAMP re-織CREB信號(hào)傳導(dǎo)通路。sponse element-binding protein and c-Fos in the hip.CREB參與成癮和學(xué)習(xí)記憶的形成,但還不能and dorsal striatum[J]. J Neurosci, 2003, 23(8)3547-3554僅用CREB解釋行為改變的長(zhǎng)久性。藥物抑制和經(jīng)m1譚蕾,羅愛林,王金韜,等氯胺酮對(duì)大鼠海馬神經(jīng)元驗(yàn)誘導(dǎo)的CREB活性相對(duì)短暫,且注射pCRE只能CREB磷酸化水平的影響J中華麻醉學(xué)雜志,2008部分促進(jìn)突觸的刺激。這表明成癮和記憶的長(zhǎng)時(shí)轉(zhuǎn)7637-639變可能啟動(dòng)更加穩(wěn)定調(diào)節(jié)機(jī)制,有其他因子的參與。12] Pandey so., Mittal n. Lumeng L, et al. Involvement of the對(duì)于精神活性物質(zhì)的成癮仍需要進(jìn)一步的實(shí)驗(yàn)研cyclic AMP-responsive element binding protein gene tran-究,為臨床戒斷和脫毒提供理論數(shù)據(jù)支持。scription factor in genetic preference for alcohol drinkingbehavior[J]. Alcohol Clin Exp Res, 1999, 23(9): 1425[參考文獻(xiàn)][1] Morgan CJ, Riccelli M, Maitland CH, et al. Long-term ef- [13] Goulart BK, de Lima MN, de Farias CB, et al. Ketaminfects of ketamine: evidence for a persisting impairment ofimpairs recognition memory consolidation and preventssource memory in recreational users[J. Drug Alcohol Delearning-induced increase in hippocampal brain-derivedpend,2004,75(3):301-308eurotrophic factor levels[J]. Neuroscience, 2010, 167(4)[2 Wang JH. Fu Y, Wilson FA, et al. Ketamine affects memo-969973ry consolidation: differential effects in T- maze and passive14]林林,黃陳平,方周溪,等.多次氯胺酮麻醉對(duì)大鼠學(xué)習(xí)avoidance paradigms in mice[J]. Neuroscience, 2006, 140記憶的影響!安徽醫(yī)藥,2006,10(7)-490-4913):993-1002[151 Ron D Signaling cascades regulating NMDA receptor sen-[3 Kinney JW, Sanchez-Alavez M, Barr AM, et al. Impairmentitivity to ethanol[J. Neuroscientist, 2004, 10(4 325-336of memory consolidation by galanin correlates with in vivo [16] Sakai R, Ukai W, Sohma H, et al. Attenuation of braininhibition of both LTP and CrEB phosphorylation[J]. Neu-derived neurotrophic factor(BDNF) by ethanol and cytorobiol Learn Mem, 2009, 92(3): 429-4fect of exogenous BDNF against ethanol dam4]王圣平,尚偉芬,宋潔,等.連續(xù)給乙醇及重復(fù)電體克對(duì)ge in neuronal cells[J]. 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(4Wnt5a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*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Wn5a的動(dòng)態(tài)表達(dá)變化及其意義。方法:將48只大鼠背部浸人恒溫水浴盆(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:在燙傷組和治療組Wnt5a表達(dá)均有顯著表達(dá),對(duì)照組有少量表達(dá)(P<0.05),傷后6-12hWn5a的表達(dá)持續(xù)升高,燙傷組12-24h開始表達(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明顯,wnt5a可能參與了燒傷膿毒癥的發(fā)生發(fā)展過程。胰島素能減少其表達(dá),可能是其保護(hù)腸黏膜機(jī)制的因素之一鍵詞]燒傷;腸黏膜;Wnt5a;大鼠「中圖分類號(hào)1R644「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 Uniity, Nantong 226001)[Abstract] Objective: To investigate the changes of the expressions of Wnt5a in intestinal mucosa of rats during the earlyage 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/kgsulin after scald injury. The expression of Wnt5a in the intestinal mucosa was detected with immunohistochemistry methodsnd 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 theIKey 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,主要是依賴于其腸道屏障的功腸潰瘍,甚至岀現(xiàn)消化道大岀血或穿孔等并發(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,不少研究資料②顯示內(nèi)最大的“內(nèi)毒素庫”。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)類科研課題(117017)*[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@126cm1005-1013for addiction[J]. Brain Res, 2011, 1406: 59-64[17 Kumar D, Deb L, Chakraborty J, et al. A polymorphism of收稿日期]2013-03-10the CreB binding protein(CrEBBP) gene is a risk fac