席青松,朱麗霞,王萍,王晶,陳華先,王芳,張瓊,王偉#

華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院a.腫瘤科,b.婦產(chǎn)科,c.神經(jīng)科,武漢 430030

大鼠局灶性腦缺血后星形膠質(zhì)細(xì)胞Cyclin D1及Cyclin E的表達(dá)*

席青松a,朱麗霞b,c,王萍c,王晶c,陳華先c,王芳c,張瓊c,王偉c#

華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院a.腫瘤科,b.婦產(chǎn)科,c.神經(jīng)科,武漢 430030

目的:研究大鼠局灶性腦缺血后星形膠質(zhì)細(xì)胞細(xì)胞周期蛋白Cyclin D1及Cyclin E的表達(dá)差異及變化。方法:建立大鼠大腦中動(dòng)脈閉塞模型,雙光源流式細(xì)胞術(shù)檢測(cè)假手術(shù)組及缺血再灌注后1 d、3 d、7 d、15 d組大鼠大腦皮質(zhì)星形膠質(zhì)細(xì)胞Cyclin D1及Cyclin E的表達(dá)。結(jié)果:假手術(shù)組大鼠星形膠質(zhì)細(xì)胞表達(dá)Cyclin D1及Cyclin E,Cyclin E的表達(dá)高于 Cyclin D1(P＜0.05);局灶性腦缺血后,星形膠質(zhì)細(xì)胞Cyclin D1的表達(dá)逐漸增加,7 d及15 d時(shí)高于假手術(shù)組(P＜0.05);局灶性腦缺血后,星形膠質(zhì)細(xì)胞Cyclin E的表達(dá)無(wú)明顯變化,各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論:局灶性腦缺血后星形膠質(zhì)細(xì)胞Cyclin D1表達(dá)增強(qiáng),提示Cyclin D1可能參與腦缺血后星形膠質(zhì)細(xì)胞的活化增殖。

Cyclin D1;Cyclin E;腦缺血;流式細(xì)胞術(shù);星形膠質(zhì)細(xì)胞

細(xì)胞周期是細(xì)胞生命活動(dòng)的基本過(guò)程,細(xì)胞在其周期時(shí)相的演化過(guò)程中經(jīng)歷增殖、分化、衰老和死亡等生理現(xiàn)象,越來(lái)越多的證據(jù)表明缺血性腦損傷后存在細(xì)胞周期的異常激活[1,2]。星形膠質(zhì)細(xì)胞是腦組織中存在的數(shù)量最多的細(xì)胞,屬可增殖細(xì)胞,缺血、損傷等刺激可激活星形膠質(zhì)細(xì)胞進(jìn)入快速增殖。本研究利用雙光源流式細(xì)胞技術(shù)觀察局灶性腦缺血后大鼠星形膠質(zhì)細(xì)胞細(xì)胞周期蛋白(Cyclin)D1、E的表達(dá),為特定細(xì)胞細(xì)胞周期調(diào)控的進(jìn)一步研究提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 材料 ①動(dòng)物:成年雄性SD大鼠25只,體質(zhì)量200～220 g,由華中科技大學(xué)同濟(jì)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。②主要試劑與材料:膠質(zhì)原纖維酸性蛋白(glial fibrillary acidic protein,GFAP)小鼠單克隆抗體、Cyclin D1及Cyclin E兔多克隆抗體(購(gòu)于NeoMarkers公司);異硫氰酸熒光素(FITC)標(biāo)記的羊抗小鼠IgG(購(gòu)于Pierce公司);R-藻紅蛋白(RPE)標(biāo)記的羊抗兔IgG抗體(購(gòu)于Proteintech Group Inc公司);其它化學(xué)試劑均為國(guó)產(chǎn)分析純?cè)噭?FACSort型流式細(xì)胞儀(購(gòu)于Becton Dickinson公司)。

1.2 方法

1.2.1 局灶性腦缺血再灌注模型制備 25只SD大鼠隨機(jī)分為(缺血再灌注后)1 d、3 d、7 d、15 d組和假手術(shù)組,每組各5只。線栓法制作大鼠大腦中動(dòng)脈閉塞(midd le cerebral artery occlusion,MCAO)模型[3]。6%水合氯醛(300 mg/kg)腹腔注射麻醉大鼠后仰臥位固定于手術(shù)臺(tái),常規(guī)消毒,頸部正中切開,分離右側(cè)頸總動(dòng)脈、頸外動(dòng)脈、頸內(nèi)動(dòng)脈,結(jié)扎并游離頸外動(dòng)脈主干一段,沿頸內(nèi)動(dòng)脈向下分離翼顎動(dòng)脈,在頸總動(dòng)脈近頸內(nèi)、外動(dòng)脈分叉處剪一小口,將涂以硅膠的4-0絲線(長(zhǎng)30mm)插入頸內(nèi)動(dòng)脈,深度為距頸總動(dòng)脈分叉處18～20 mm,栓塞右側(cè)大腦中動(dòng)脈,90min后拔出絲線,縫合皮膚,術(shù)中維持動(dòng)物肛溫(36.6±0.5)℃;假手術(shù)組不插入線栓。蘇醒后出現(xiàn)右前肢屈曲及前進(jìn)時(shí)向右側(cè)轉(zhuǎn)圈者,為造模成功大鼠。

1.2.2 雙光源流式細(xì)胞術(shù)(flow cy tometry,FCM)檢測(cè)星形膠質(zhì)細(xì)胞Cyclin D1及Cyclin E的表達(dá)

假手術(shù)組術(shù)后1 d取腦,其它各組分別于術(shù)后1 d、3 d、7 d、15 d取腦。深度麻醉大鼠,冰上快速剝離大腦,取缺血側(cè)梗死邊緣區(qū)皮質(zhì),機(jī)械法制成單細(xì)胞懸液,200目篩網(wǎng)過(guò)濾,PBS洗滌、離心2次,5×106/管分裝,加入75%冷乙醇固定細(xì)胞過(guò)夜;PBS洗滌、離心 2次,含 0.25%TritonX-100的 PBS冰上預(yù)處理5 min,離心,棄 TritonX-100后加PBS洗滌2次,離心,分別加入GFAP抗體(1∶300)和Cyclin D1抗體(1∶100)及GFAP抗體(1∶300)和Cyclin E抗體(1∶100),4℃孵育過(guò)夜;PBS洗滌、離心3次,加入FITC標(biāo)記的羊抗小鼠IgG(1∶100)和R-PE標(biāo)記的羊抗兔IgG抗體(1∶100),室溫避光孵育60min,PBS洗滌3次,流式細(xì)胞儀檢測(cè)。檢測(cè)時(shí)設(shè)陰性對(duì)照(以2%BSA代替一抗),激發(fā)波長(zhǎng)488 nm。Cellquest軟件分別對(duì)所測(cè)GFAP陽(yáng)性表達(dá)細(xì)胞Cyclin D1及Cyclin E的表達(dá)率進(jìn)行雙參數(shù)檢測(cè),計(jì)算陽(yáng)性細(xì)胞百分率。

1.3 統(tǒng)計(jì)學(xué)處理 用SPSS 12.0統(tǒng)計(jì)軟件處理數(shù)據(jù),計(jì)量資料以(x±s)表示,F檢驗(yàn),P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

FCM檢測(cè)結(jié)果顯示,假手術(shù)組大鼠大腦皮質(zhì)星形膠質(zhì)細(xì)胞表達(dá)Cyclin D1及Cyclin E,且Cyclin E的表達(dá)高于Cyclin D1(P＜0.05);局灶性腦缺血后隨時(shí)間增加,星形膠質(zhì)細(xì)胞Cyclin D1的表達(dá)逐漸增加,7 d及15 d時(shí)高于假手術(shù)組(P＜0.05);局灶性腦缺血后,星形膠質(zhì)細(xì)胞Cyclin E的表達(dá)無(wú)明顯變化,各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義,見(jiàn)圖1A-E及表1。

3 討論

中樞神經(jīng)系統(tǒng)中存在大量星形膠質(zhì)細(xì)胞,為神經(jīng)元提供代謝和營(yíng)養(yǎng)支持,在保護(hù)神經(jīng)元存活、調(diào)節(jié)突觸功能及神經(jīng)再生和修復(fù)中起重要作用[4,5]。研究表明腦缺血損傷導(dǎo)致星形膠質(zhì)細(xì)胞大量增殖,最終形成膠質(zhì)瘢痕,阻礙神經(jīng)修復(fù)和軸突再生[6]?？刂颇X缺血后異常激活的星形膠質(zhì)細(xì)胞的細(xì)胞周期,減少膠質(zhì)疤痕形成,促進(jìn)神經(jīng)軸突再生,是腦缺血后神經(jīng)功能恢復(fù)研究的熱點(diǎn)。

圖1 A-E 各組星形膠質(zhì)細(xì)胞Cyclin D1及Cyclin E FMC檢測(cè)散點(diǎn)圖 A:假手術(shù)組;B:1 d組;C:3 d組;D:7 d組;E:15 d組;散點(diǎn)圖橫線以上代表Cyclin D1、Cyclin E陽(yáng)性細(xì)胞群,橫線以下代表Cyclin D1、Cyclin E陰性細(xì)胞群

表1 各組星形膠質(zhì)細(xì)胞Cyclin D1及Cyclin E陽(yáng)性細(xì)胞百分率比較(x-±s,%)

細(xì)胞周期指由細(xì)胞分裂結(jié)束到下一次細(xì)胞分裂結(jié)束所經(jīng)歷的過(guò)程,分為G1、S、G2及 M 期。細(xì)胞周期調(diào)控受多種生化因素相互影響,其核心機(jī)制是Cyclin的時(shí)相性起伏及細(xì)胞周期蛋白依賴性激酶(Cyclin dependent kinase,CDK)的序列激活[7]。Cyclin/CDK的作用具有時(shí)相特異性和細(xì)胞特異性,在不同神經(jīng)細(xì)胞類型及細(xì)胞周期的不同階段,起關(guān)鍵作用的Cyclin/CDK分子不同[8,9]。若能找出調(diào)控星形膠質(zhì)細(xì)胞增殖的特定蛋白,并對(duì)其定向干預(yù),將有助于抑制膠質(zhì)疤痕形成,促進(jìn)軸突再生。

細(xì)胞周期啟動(dòng)的主要調(diào)控點(diǎn)在G1期,是細(xì)胞唯一能接受外界增殖或抑制信號(hào)的時(shí)期。Gl期主要的細(xì)胞周期調(diào)控蛋白為Cyclin D 1和Cyclin E,分別與其相應(yīng)的CDK結(jié)合形成Cyclin-CDK復(fù)合物(CyclinDl-CDK 4/6,Cyclin E-CDK 2),是G1/S 期轉(zhuǎn)化的關(guān)鍵時(shí)期[10]。本實(shí)驗(yàn)發(fā)現(xiàn)局灶性腦缺血后星形膠質(zhì)細(xì)胞Cyclin D1的表達(dá)逐漸增加,7 d及15 d時(shí)高于假手術(shù)組,Cyclin E的表達(dá)無(wú)明顯變化,推測(cè)在星形膠質(zhì)細(xì)胞增殖、細(xì)胞周期由G1期向S期過(guò)渡的轉(zhuǎn)變中,Cyclin D 1發(fā)揮更為重要的作用,控制星形膠質(zhì)細(xì)胞細(xì)胞周期進(jìn)程,促進(jìn)星形膠質(zhì)細(xì)胞的增生。已有實(shí)驗(yàn)證實(shí),Cyclin D1基因敲除對(duì)星形膠質(zhì)細(xì)胞的增殖有明顯抑制作用[11]。Cyclin E在星形膠質(zhì)細(xì)胞中的表達(dá)強(qiáng)于Cyclin D1,其原因及作用還需要進(jìn)一步研究。

[1]Liu DX,G reene LA.Regu lation of Neuronal Survival and Death by E2F-dependent Gene Repression and Derepression[J].Neuron(S0896-6273),2001,32(3):425-438.

[2]Love S.Neuronal Expression of CellCycle-related Proteins after Brain Ischaem ia in M an[J].Neuroscience Letters(S0304-3940),2003,353(1):29-32.

[3]Longa EZ,Weinstein PR,Carlson S,etal.Reversible Middle Cerebral A rtery Occlusion Without Craniectomy in Rats[J].Stroke(S0039-2499),1989,20(1):84-91.

[4]Aschner M,Sonnew ald U,Tan KH.Astrocy te Modulation of Neurotoxic Injury[J].Brain Pathol(S1015-6305),2002,12(4):475-481.

[5]Swanson RA,Ying W,Kauppinen TM.Astrocyte Influences on Ischemic Neuronal Death[J].Curr M ol M ed(S1566-5240),2004,4(2):193-205.

[6]Kernie SG,Erwin TM,Parada LF.Brain Remodeling Due to Neuronal and Astrocytic Proliferation after Controlled Cortical In jury in mice[J].JNeurosci Res(S0360-4012),2001,66(3):317-326.

[7]Bohnsack BL,H irschi KK.Nutrient Regu lation of Cell Cycle Progression[J].Annu Rev Nutr(S0199-9885),2004,24:433-453.

[8]Ross ME.Cell Division and the Nervous System:Regulating the Cycle from Neural Differentiation to Death[J].T rends Neurosci(S0166-2236),1996,19(2):62-68.

[9]Ohnuma S,Philpott A,H arris WA.Cell Cycle and Cell Fate in the Nervous System[J].Curr Opin Neurobiol(S0959-4388),2001,11(1):66-73.

[10]Donnellan R,Chesty R.Cyclin D1 and Human Neop lasia[J].Mo l Pathol(S1366-8714),1998,51(1):1-7.

[11]Zhu Z,Zhang Q,Yu Z,et al.Inhibiting Cell Cycle Progression Reduces Reactive Astrog liosis Initiated by Scratch In jury in vitro and by Cerebral Ischem ia in vivo[J].G lia(S0894-1491),2007,55(5):546-558.

Exp ressions of Cyclin D1 and Cyclin E in Astrocytes of Rats after Focal Cerebral Ischem ia

X IQing-song▲,ZHU Lixia,WANG Ping,WANG Jing,CHEN Hua-xian,W ANG Fang,ZHANGQiong,WANGWei.▲Department of Oncology,Tong ji Hospital,Tong ji Medica l College,H uazhong University of Science and Technology,Wuhan 430030,China

Objective:To investigate the change of the expression of Cyclin D1 and Cyclin E in astrocy tes of rats after focal cerebral ischem ia.Methods:Ischem ia w as induced by temporary m idd le cerebral artery occlusion(MCAO).The rats su ffered from MCAO were sacrificed on days 1,3,7 or 15 after reperfusion and rats in control group w ere sacrificed on day 1 after surgical operation.The brain w as taken and the expressions of Cyclin D1 and Cyclin E in astrocy tes were detected by flow cy tometry.Resu lts:The exp ressions of Cyclin D1 and Cyclin E were detected in astrocy tes of the controls and the exp ression of Cyclin E w as higher than that of Cyclin D1(P＜0.05).After focal cerebral ischem ia,the expression of Cyclin D1 in astrocytesw as significantly increased on the edge of ischemic core at7 and 15 days after reperfusion(P＜0.05),while the exp ression of Cyclin E had no significant variation.Conclusion:Cyclin D1 may take part in the p rogression of the cell cycle in G1/S transition in p roliferating astrocytes.

Cyclin D 1;Cyclin E;cerebral ischemia;flow cytometry;astrocy tes

R741;R741.02

A

1001-117X(2010)01-0014-03

10.3870/sjsscj.2010.01.005

國(guó)家杰出青年基金(No.30725019)

2009-11-19

#【通訊作者】王偉,Tel:86-27-83663657,E-mail:wwang_tjh@yahoo.com.cn。