李建立,梁巍,吳紅海,侯艷寧

(1.河北省人民醫(yī)院麻醉科,石家莊 050051;2.河北省人民醫(yī)院小兒外科,石家莊 050051;3.白求恩國際和平醫(yī)院藥劑科,石家莊 050082)

17β雌二醇對(duì)氯胺酮所致神經(jīng)元凋亡的影響

李建立1,梁巍2,吳紅海3,侯艷寧3

(1.河北省人民醫(yī)院麻醉科,石家莊 050051;2.河北省人民醫(yī)院小兒外科,石家莊 050051;3.白求恩國際和平醫(yī)院藥劑科,石家莊 050082)

目的 研究17β雌二醇對(duì)氯胺酮所致原代培養(yǎng)皮質(zhì)神經(jīng)元凋亡的影響及其機(jī)制。方法原代培養(yǎng)皮質(zhì)神經(jīng)元,體外培養(yǎng)7 d,隨機(jī)分為空白對(duì)照組(給予相同體積的DMSO),雌二醇組(17β雌二醇終濃度為0.1 μmol·L-1),氯胺酮組(氯胺酮終濃度為100 μmol·L-1),氯胺酮+雌二醇組(氯胺酮,17β雌二醇終濃度分別為100 μmol·L-1,0.1 μmol·L-1)。噻唑藍(lán)(MTT)法檢測(cè)神經(jīng)元存活率,Hoechest33258染色法檢測(cè)皮質(zhì)神經(jīng)元凋亡,Western-blot法測(cè)定cleaved-Caspase-3及Bcl-2蛋白表達(dá)。結(jié)果氯胺酮組神經(jīng)元存活率(54.02±7.78)%,明顯低于空白對(duì)照組;氯胺酮+雌二醇組神經(jīng)元存活率(88.09±6.54)%,明顯高于氯胺酮組。神經(jīng)元經(jīng)Hoechest33258染色在熒光顯微鏡下觀察,氯胺酮組神經(jīng)元凋亡[凋亡率(49.50±4.34)%]較空白對(duì)照組明顯增加,氯胺酮+雌二醇組[凋亡率(15.74±3.40)%]較氯胺酮組神經(jīng)元凋亡下降。氯胺酮組cleaved-Caspase-3表達(dá)明顯增加,Bcl-2明顯下降,而氯胺酮+雌二醇組較氯胺酮組cleaved-Caspase-3表達(dá)明顯下降,Bcl-2表達(dá)明顯升高。結(jié)論17β雌二醇通過抑制神經(jīng)元凋亡對(duì)抗氯胺酮誘導(dǎo)的皮質(zhì)神經(jīng)元損傷,產(chǎn)生保護(hù)作用。

17β雌二醇;氯胺酮;皮質(zhì)神經(jīng)元;凋亡

氯胺酮是一種N-甲基-D-天冬氨酸受體(N-methyl-D-aspartic acid receptor,NMDAR)拮抗藥。最近的研究表明,在中樞神經(jīng)系統(tǒng)快速發(fā)育期反復(fù)應(yīng)用氯胺酮可影響神經(jīng)系統(tǒng)的發(fā)育,導(dǎo)致神經(jīng)元凋亡增加,甚至影響成年后的學(xué)習(xí)記憶能力[1]。目前,大量的研究表明,氯胺酮可導(dǎo)致原代培養(yǎng)的皮質(zhì)神經(jīng)元凋亡[2-3]。因此,對(duì)于氯胺酮引起的發(fā)育期大腦損傷,尋找有效的預(yù)防治療措施已成為麻醉醫(yī)師關(guān)注的重要課題。17β雌二醇為一種神經(jīng)活性甾體,不僅參與調(diào)節(jié)神經(jīng)系統(tǒng)的發(fā)育,還影響神經(jīng)系統(tǒng)的功能,發(fā)揮神經(jīng)保護(hù)作用,該藥對(duì)神經(jīng)系統(tǒng)的保護(hù)作用及機(jī)制已成為國內(nèi)外研究的熱點(diǎn)[4]。17β雌二醇對(duì)氯胺酮引起的皮質(zhì)神經(jīng)元凋亡是否有保護(hù)作用以及其可能的機(jī)制,筆者未見文獻(xiàn)報(bào)道。本實(shí)驗(yàn)從細(xì)胞凋亡的形態(tài)學(xué)變化及與凋亡相關(guān)蛋白cleaved-caspase-3及Bcl-2表達(dá)變化等方面研究17β雌二醇對(duì)氯胺酮導(dǎo)致原代培養(yǎng)皮質(zhì)神經(jīng)元凋亡的影響。

1 材料與方法

1.1 藥物及試劑 氯胺酮注射液(福建古田藥業(yè)有限公司,規(guī)格:100 mg/2 mL,批號(hào):H35020148),達(dá)爾伯克改良伊格爾培養(yǎng)液(Dulbecco's modification of Eagle'smedium,DMEM,批號(hào):11965-092)、Neurobasal培養(yǎng)液(批號(hào):21103049)、B27促生長劑(批號(hào):17504)購自美國Gibco公司,17β雌二醇(規(guī)格:1 mg,批號(hào):E8875)、二甲亞砜(dimethyl sulfoxide, DMSO,批號(hào):D2650)、噻唑藍(lán)(thiazole blue,MTT,批號(hào):M5655)均購自美國Sigma公司。Hoechest33258 (批號(hào):23491-45-4)與胰蛋白酶(批號(hào):T1320)購自北京索來寶公司。cleaved-Caspase-3(批號(hào):9661S)和Bcl-2(批號(hào):6516S)抗體購自美國Cell signal Technology公司。

1.2 皮質(zhì)神經(jīng)元原代培養(yǎng) 參照文獻(xiàn)[5]方法,略加改進(jìn)。取新生24 h內(nèi)的SD幼鼠[清潔級(jí),體質(zhì)量5～6 g,雌雄不限,河北省實(shí)驗(yàn)動(dòng)物中心提供,生產(chǎn)許可證號(hào):SCXK(冀)2008-1003,合格證號(hào):1306107],在無菌操作下迅速取出大腦,用D-Hanks液清洗后取出大腦皮質(zhì),剪成約1 mm×1 mm×1 mm大小組織塊,經(jīng)0. 125%胰蛋白酶37℃消化15 min,用含10%胎牛血清的DMEM培養(yǎng)液終止消化,把細(xì)胞轉(zhuǎn)移到含10%胎牛血清的DMEM培養(yǎng)液中制成細(xì)胞懸液,經(jīng)100目鋼絲篩(篩孔內(nèi)徑0.149 mm)過濾,計(jì)數(shù)后按1×106·mL-1密度接種于經(jīng)多聚賴氨酸處理的培養(yǎng)板,37℃、5%二氧化碳(CO2)培養(yǎng)箱內(nèi)培養(yǎng)24 h后全量換neurobasal +B27培養(yǎng)液,以后每隔2 d半量換液1次。體外培養(yǎng)7 d的神經(jīng)元用于實(shí)驗(yàn)。

1.3 實(shí)驗(yàn)分組 隨機(jī)分為空白對(duì)照組(給予相同體積的DMSO),雌二醇組(17β雌二醇終濃度為0.1 μmol·L-1),氯胺酮組(氯胺酮終濃度為100 μmol·L-1),氯胺酮+雌二醇組(氯胺酮,17β雌二醇終濃度分別為100 μmol·L-1,0.1 μmol·L-1)。

1.4 MTT法檢測(cè)神經(jīng)元存活率 將細(xì)胞接種于96孔板,體外培養(yǎng)至第7天,分別加入不同的藥物處理24 h,翻板法棄去培養(yǎng)液,每孔加入MTT液10 μL, 37℃孵育4 h,棄去上清液,加入DMSO 200 μL,輕輕振蕩溶解甲瓚結(jié)晶,在多功能酶標(biāo)儀上測(cè)定570 nm波長處的吸光度,以對(duì)照組平均吸收值為100%,以各處理組吸收值與對(duì)照組的比值計(jì)算存活率。存活率(%)=實(shí)驗(yàn)組吸光度/對(duì)照組吸光度×100%。

1.5 Hoechest33258核染色法檢測(cè)神經(jīng)元凋亡 將神經(jīng)元接種于6孔培養(yǎng)板,按上述方法培養(yǎng),分組處理后,移去培養(yǎng)液,用4℃磷酸鹽緩沖液(phosphate buffer solution,PBS)沖洗1遍,加入4%多聚甲醛固定30 min,棄固定液,用冷PBS液沖洗3遍,加入Hoechest33258染色8 min,PBS液漂洗2遍,熒光顯微鏡下隨機(jī)選取5個(gè)視野進(jìn)行形態(tài)學(xué)觀察和計(jì)數(shù),計(jì)算凋亡率。凋亡率(%)=凋亡細(xì)胞數(shù)/細(xì)胞總數(shù)× 100%。

1.6 Western-blot法測(cè)定cleaved-Caspase-3和Bcl-2蛋白表達(dá) 細(xì)胞經(jīng)處理后,收集細(xì)胞,裂解液裂解細(xì)胞提取細(xì)胞總蛋白,二喹啉甲酸(bicinchoninic acid, BCA)法檢測(cè)樣品蛋白含量。取待測(cè)蛋白質(zhì)50 μg加上樣緩沖液煮沸變性,于10%十二烷基硫酸鈉-聚丙烯酰胺凝膠中100 V電泳1.5 h,轉(zhuǎn)膜1 h,加入cleaved-Caspase-3和Bcl-2抗體(1∶2 000),4℃過夜,常規(guī)洗滌,加羊抗鼠二抗(1∶5 000)37℃孵育60 min,洗滌,電化學(xué)法發(fā)光、顯影、掃描,用凝膠圖像處理系統(tǒng)分析目標(biāo)條帶與內(nèi)參條帶吸光度的比值。實(shí)驗(yàn)重復(fù)3次,設(shè)β-actin蛋白為內(nèi)參。

2 結(jié)果

2.1 17β雌二醇處理對(duì)氯胺酮誘導(dǎo)的皮質(zhì)神經(jīng)元損傷保護(hù)作用 空白對(duì)照組神經(jīng)元存活率為[(99.98± 5.76)%],與空白對(duì)照組比較,雌二醇組神經(jīng)元存活率[(99.85±8.43)%]未見明顯變化,氯胺酮組神經(jīng)元存活率[(54.02±7.78)%]明顯下降(P＜0.01)。與氯胺酮組比較,氯胺酮+雌二醇組神經(jīng)元存活率[(88.09±6.54)%]明顯增加(P＜0.01)。圖1。

2.2 17β雌二醇處理對(duì)氯胺酮誘導(dǎo)皮質(zhì)神經(jīng)元凋亡的影響 經(jīng)Hoechest33258染色熒光在顯微鏡下觀察,空白對(duì)照組與雌二醇組細(xì)胞核內(nèi)呈均勻分布的淡藍(lán)色熒光,內(nèi)有較深的藍(lán)色顆粒,有少量凋亡細(xì)胞呈亮藍(lán)色,凋亡率(5.15±0.21)%。氯胺酮組凋亡細(xì)胞明顯增加(P＜0.01),大多細(xì)胞呈亮藍(lán)色,部分細(xì)胞核呈碎片狀,部分染色質(zhì)邊集,凋亡率(49.50±4.34)%。氯胺酮+雌二醇組神經(jīng)元凋亡數(shù)量較氯胺酮組明顯減少(P＜0.01),凋亡率為(15.74±3.40)%。見圖2,圖3。

與空白對(duì)照組比較,*1P＜0.01;與氯胺酮組比較,*2P＜0.01圖1 不同處理對(duì)神經(jīng)元存活率的影響Compared with blank control group,*1P＜0.01;compared with ketamine group,*2P＜0.01Fig.1 Effect of different treatments on neuron viability

2.3 17β雌二醇處理對(duì)氯胺酮誘導(dǎo)皮質(zhì)神經(jīng)元凋亡時(shí)cleaved-Caspase-3表達(dá)的影響 采用Western-blot法檢測(cè),空白對(duì)照組神經(jīng)元cleaved-Caspase-3水平為(0.56±0.02)。雌二醇組為(0.58±0.03),與空白對(duì)照組比較未見明顯變化。氯胺酮組為(1.24±0.08),較對(duì)照組明顯增加(P＜0.01)。氯胺酮+雌二醇組神經(jīng)元cleaved-Caspase-3水平為(0.78±0.10),較氯胺酮組明顯下降(P＜0.01)。見圖4,5。

2.4 17β雌二醇處理對(duì)氯胺酮誘導(dǎo)皮質(zhì)神經(jīng)元凋亡時(shí)Bcl-2表達(dá)的影響 采用Western-blot法檢測(cè),空白對(duì)照組神經(jīng)元Bcl-2水平為(1.12±0.1),雌二醇組為(1.16± 0.2),與空白對(duì)照組比較,未見明顯變化。氯胺酮組神經(jīng)元Bcl-2水平為(0.58±0.04),較對(duì)照組明顯降低(P＜0.01)。氯胺酮+雌二醇組神經(jīng)元Bcl-2水平為(0.84± 0.08),較氯胺酮組明顯增加(P＜0.01)。見圖6,7。

3 討論

A.空白對(duì)照組;B.雌二醇組;C.氯胺酮組;D.氯胺酮+雌二醇組圖2 不同處理對(duì)神經(jīng)元凋亡的影響(×100)A.blank control group;B.estradiol group;C.ketamine group;D.ketamine plus estradiol groupFig.2 Effect of different treatments on neuron apoptosis(×100)

與空白對(duì)照組比較,*1P＜0.01;與氯胺酮組比較,*2P＜0.01圖3 4組神經(jīng)元凋亡率測(cè)定結(jié)果Compared with blank control group,*1P＜0.01;compared with ketamine group,*2P＜0.01Fig.3 Detection results of neuron apoptosis in four groups of neurons

圖4 不同處理對(duì)神經(jīng)元Cleaved-caspase-3表達(dá)的影響Fig.4 Effect of different treatments on expression of Cleaved-caspase-3

氯胺酮自1965年用于臨床以來,在臨床麻醉中一直享有獨(dú)特的地位,廣泛用于兒童麻醉。但是IKONOMIDOU等[6]研究發(fā)現(xiàn),出生14 d內(nèi)的新生大鼠,使用NMDAR拮抗藥MK-801后能引起廣泛的神經(jīng)細(xì)胞凋亡變性,成年大鼠卻沒有出現(xiàn)此現(xiàn)象,這說明在大鼠腦發(fā)育高峰期(出生前l(fā) d至出生后14 d),使用NMDAR拮抗藥會(huì)干擾中樞神經(jīng)系統(tǒng)發(fā)育。氯胺酮是NMDAR拮抗藥,近年來大量研究證明氯胺酮對(duì)發(fā)育期大腦和原代培養(yǎng)的皮質(zhì)神經(jīng)元產(chǎn)生損傷[1-3]。因此尋找安全有效的預(yù)防治療措施來減少發(fā)育期氯胺酮的神經(jīng)毒性,顯得尤為重要。

與空白對(duì)照組比較,*1P＜0.01;與氯胺酮組比較,*2P＜0.01圖5 4組神經(jīng)元Cleaved-caspase-3表達(dá)測(cè)定結(jié)果Compared with blank control group,*1P＜0.01;compared with ketamine group,*2P＜0.01Fig.5 Detection on Cleaved-caspase-3 expression in four groups of neurons

圖6 不同處理對(duì)神經(jīng)元Bcl-2表達(dá)的影響Fig.6 Effect of different treatments on expression of Bcl-2

圖7 4組神經(jīng)元Bcl-2表達(dá)測(cè)定結(jié)果Fig.7 Detection on Bcl-2 expression in four groups of neurons

已有研究發(fā)現(xiàn),鋰、促紅細(xì)胞生成素、可樂定等可對(duì)氯胺酮引起的神經(jīng)損傷產(chǎn)生保護(hù)作用[7-9],但它們?cè)趮胗變簯?yīng)用的安全性需要進(jìn)一步研究。17β雌二醇為一種神經(jīng)活性甾體,目前大量的研究證實(shí)其具有神經(jīng)保護(hù)作用,如對(duì)老年癡呆、缺血缺氧性損傷、興奮性毒性損傷以及氧化應(yīng)激等損傷產(chǎn)生保護(hù)作用[10]。另外有研究報(bào)道,17β雌二醇可對(duì)NMDA受體拮抗劑MK-801誘導(dǎo)的發(fā)育期大腦凋亡樣損傷以及由麻醉藥異氟醚、氧化亞氮、咪達(dá)唑侖聯(lián)合應(yīng)用引起的發(fā)育期大腦損傷產(chǎn)生保護(hù)作用[11-12]。然而17β雌二醇是否對(duì)氯胺酮引起的發(fā)育期大腦損傷產(chǎn)生保護(hù)作用以及機(jī)制,國內(nèi)外未見報(bào)道。

目前研究認(rèn)為凋亡是氯胺酮引起發(fā)育期大腦損傷的重要機(jī)制[13]。熒光顯微鏡檢測(cè)法因結(jié)果可靠,易于操作而成為細(xì)胞凋亡檢測(cè)的常用手段,尤其適用于體外培養(yǎng)細(xì)胞的檢測(cè)[14]。Hoechest是一種細(xì)胞膜通透性的熒光染料,Hoechest33258是其中一種常見染料。正常細(xì)胞和中早期凋亡細(xì)胞均可被Hoechest著色。正常細(xì)胞核呈圓形,淡藍(lán)色,而凋亡的細(xì)胞核呈碎片狀,邊集的結(jié)構(gòu)特征,由于濃集而使染色增強(qiáng),熒光更為明亮,呈亮藍(lán)色。本研究采用Hoechest33258染色來觀察凋亡的神經(jīng)元,發(fā)現(xiàn)氯胺酮導(dǎo)致凋亡神經(jīng)元明顯增加,而17β雌二醇處理后會(huì)使凋亡的神經(jīng)元明顯減少。Caspase-3是各種細(xì)胞凋亡通路的最終執(zhí)行者。Bcl-2作為抗凋亡蛋白,通過抑制線粒體細(xì)胞色素C以及Caspase-3的活化而產(chǎn)生抗凋亡作用[15]。本實(shí)驗(yàn)首次對(duì)17β雌二醇處理保護(hù)原代培養(yǎng)的皮質(zhì)神經(jīng)元免受氯胺酮引起的損傷進(jìn)行了研究。結(jié)果顯示100 μmol·L-1氯胺酮使原代培養(yǎng)的皮質(zhì)神經(jīng)元存活率明顯下降,同時(shí)神經(jīng)元凋亡明顯增加,cleaved-Caspase-3表達(dá)明顯增加,抗凋亡蛋白Bcl-2表達(dá)明顯下降,17β雌二醇處理能對(duì)抗氯胺酮引起的神經(jīng)元損傷,使神經(jīng)元存活率增加,神經(jīng)元凋亡減少, cleaved-Caspase-3表達(dá)明顯下降,抗凋亡蛋白Bcl-2表達(dá)增加。本研究表明氯胺酮可通過激活神經(jīng)元凋亡途徑導(dǎo)致神經(jīng)元存活率降低,而神經(jīng)活性甾體17β雌二醇可通過抗凋亡機(jī)制來抑制氯胺酮的神經(jīng)毒性作用,維持神經(jīng)元的正常成長。

總之17β雌二醇具有保護(hù)神經(jīng)元免受氯胺酮損傷的作用,其機(jī)制是通過抗凋亡作用實(shí)現(xiàn)的。本研究為圍術(shù)期應(yīng)用17β雌二醇預(yù)防麻醉藥對(duì)嬰幼兒大腦產(chǎn)生神經(jīng)損傷提供了初步的實(shí)驗(yàn)依據(jù)。

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DOI 10.3870/yydb.2014.11.009

Effects of 17β-estradiol on Ketamine-induced Neuroapoptosis

LI Jian-li1,LIANG Wei2,WU Hong-hai3,HOU Yan-ning3
(1.Department of Anesthesiology,Hebei General Hospital,Shijiazhuang 050051,China;2.Department of Pediatric Surgery,Hebei General Hospital,Shijiazhuang 050051,China;3.Department of Pharmacy,Bethune International Peace Hospital of Chinese PLA,Shijiazhuang 050082,China)

ObjectiveTo investigate the protective effect and the mechanisms of 17β-estradiol on ketamine-induced apoptosis on primary cultured rat cortical neurons.MethodsCortical neurons were primarily cultured for seven days,then divided into four groups:control group(treated with equal valume of DMSO),estradiol-treated group(treated with 0.1 μmol·L-117β-estradiol),ketamine-treated group(treated with 100 μmol·L-1ketamine),ketamine plus 17β-estradioltreated group(treated with 0.1 μmol·L-117β-estradiol+100μmol·L-1ketamine).The neurons were treated for 24 hours.The neuron viability was determined by MTT.Neuroapoptosis was measured by nuclear morphometry after Hoechest 33258 dying. Western blotting was performed to detect the expression levels of cleaved-caspase-3 and Bcl-2protein.ResultsThe neuron viability in the ketamine group was(54.02±7.78)%,significantly decreased from the control group,whereas ketamine plus 17βestradiol increased the cell viability to(88.09±6.54)%,significantly higher than the ketamine group.The neuroapoptosis rate in the ketamine group was(49.50±4.34)%,significantly increased from the control group,while that in the drug combination group was(15.74±3.40)%,significantly lower compared with the ketamine group.Meanwhile,the cleaved-caspase-3 expression increased,and Bcl-2 expression decreased remarkably after ketamine treatment,while which was reversed in the drug combination group.Conclusion17β-estradiol can protect against ketamine-induced injury by inhibiting neuron apoptosis.

17β-estradiol;Ketamine;Cortex neurons;Neuroa apoptosis

R971.2;R965

A

1004-0781(2014)11-1434-05

2013-11-12

2014-01-05

李建立(1976-),男,河北趙縣人,副主任醫(yī)師,在讀博士,主要從事麻醉藥理學(xué)研究。E-mail:hblijianli@163. com。

侯艷寧(1957-),女,河北高陽人,教授,博士生導(dǎo)師,博士,主要從事神經(jīng)藥理學(xué)研究。電話:0311-87978503, E-mail:biph2011@163.com。