沈夏鋒，田 閃，吳 毅，朱玉連

跑臺(tái)運(yùn)動(dòng)對(duì)腦外傷大鼠基質(zhì)金屬蛋白酶-2表達(dá)的影響①

沈夏鋒1，田 閃2，吳 毅2，朱玉連2

目的觀察跑臺(tái)訓(xùn)練對(duì)腦外傷大鼠基質(zhì)金屬蛋白酶-2(MMP-2)表達(dá)的影響。方法55只成年Sprague-Dawley大鼠隨機(jī)分為假手術(shù)組(n=15)、對(duì)照組(n=20)和運(yùn)動(dòng)組(n=20)，后兩組復(fù)制中重度腦外傷模型，運(yùn)動(dòng)組進(jìn)行電動(dòng)跑臺(tái)訓(xùn)練1周。術(shù)后6、12、18、24 d，采用足誤試驗(yàn)評(píng)定運(yùn)動(dòng)協(xié)調(diào)功能；術(shù)后48 h，采用伊文思藍(lán)(EB)染色檢測(cè)大鼠血腦屏障通透性；術(shù)后7 d，Western blotting檢測(cè)MMP-2的表達(dá)。結(jié)果運(yùn)動(dòng)組在術(shù)后6 d評(píng)分明顯降低，但隨后增高，其中12 d、18 d及24 d評(píng)分比對(duì)照組高(F＞4.793, P＜0.05)；運(yùn)動(dòng)組EB滲出量顯著減少(t=-8.091,P＜0.001)；運(yùn)動(dòng)組MMP-2蛋白表達(dá)顯著下降(t=-13.12,P＜0.001)。結(jié)論大鼠腦外傷后早期進(jìn)行跑臺(tái)訓(xùn)練，可改善運(yùn)動(dòng)功能，可能與抑制MMP-2，從而減輕血腦屏障破壞有關(guān)。

腦外傷；跑臺(tái)訓(xùn)練；血腦屏障；基質(zhì)金屬蛋白酶；大鼠

腦外傷的病理生理過(guò)程包括原發(fā)性損傷和繼發(fā)性損傷[1]。原發(fā)性損傷直接由創(chuàng)傷引起，繼發(fā)性損傷持續(xù)時(shí)間較長(zhǎng)，包括腦水腫、谷氨酸興奮性中毒、脂質(zhì)過(guò)氧化、血腦屏障破壞、線粒體功能失調(diào)、炎癥、凋亡和細(xì)胞壞死等復(fù)雜的病理過(guò)程和級(jí)聯(lián)反應(yīng)[2-4]。最近研究表明，血腦屏障的破壞貫穿于腦外傷原發(fā)性和繼發(fā)性損傷，在腦外傷病理生理中占據(jù)重要地位，是腦外傷治療的新方向[5-8]。

運(yùn)動(dòng)訓(xùn)練是一組以身體鍛煉為主的治療方法，在國(guó)內(nèi)外腦外傷后康復(fù)治療中應(yīng)用廣泛，療效顯著。有研究表明，運(yùn)動(dòng)訓(xùn)練可以減輕腦外傷大鼠的神經(jīng)功能障礙，促進(jìn)運(yùn)動(dòng)能力和學(xué)習(xí)記憶能力的恢復(fù)[9-12]?；谘X屏障的完整性對(duì)腦外傷病理發(fā)展的重要意義，早期運(yùn)動(dòng)訓(xùn)練對(duì)腦損傷后血腦屏障及相關(guān)的基質(zhì)金屬蛋白酶-2(matrix metalloproteinases 2,MMP-2)的影響目前還缺乏相關(guān)研究。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組

健康成年Sprague-Dawley大鼠55只，體質(zhì)量250～270 g，由上海西普爾·必凱實(shí)驗(yàn)動(dòng)物有限公司提供，采用隨機(jī)數(shù)字表法分成假手術(shù)組15只、對(duì)照組20只和運(yùn)動(dòng)組20只。

1.2 造模方法

異氟醚吸入誘導(dǎo)麻醉，大鼠俯臥位固定于操作臺(tái)上，異氟醚全身麻醉。額頂部剃毛、消毒，沿中線矢狀線切開(kāi)皮膚，暴露顱頂骨，用牙科電鉆沿中線右旁5 mm前囟部位做直徑5 mm骨窗，暴露完整硬腦膜。使用控制性皮質(zhì)沖擊顱腦損傷儀器(PSI公司)，不銹鋼打擊頭對(duì)準(zhǔn)骨窗，速度4 m/s、深度3.2 mm撞擊，造成運(yùn)動(dòng)組和對(duì)照組大鼠中重度腦外傷[8]。小心止血、縫合。大鼠出現(xiàn)部分反射活動(dòng)消失、對(duì)側(cè)肢體功能障礙等表現(xiàn)，提示造模成功。

假手術(shù)組大鼠同法暴露硬腦膜，不進(jìn)行撞擊，直接止血和縫合傷口。

1.3 干預(yù)方法

運(yùn)動(dòng)組從術(shù)后1 d起，每天在DSPT-202電動(dòng)跑臺(tái)(立泰生物科技有限公司)上行運(yùn)動(dòng)干預(yù)，傾角0°，速度3 m/min，運(yùn)動(dòng)30 min，共1周。對(duì)照組和假手術(shù)組每天置于跑臺(tái)機(jī)上30 min，傾角0°，速度0。

1.4 觀察指標(biāo)

1.4.1 足誤試驗(yàn)

運(yùn)動(dòng)組和對(duì)照組各取12只大鼠，假手術(shù)組取7只大鼠，分別于術(shù)后6 d、12 d、18 d及24 d，參考Gerlinde介紹的操作步驟進(jìn)行測(cè)評(píng)[12-13]。0分：左前爪完全踩空，未觸到橫檔即掉下，身體失去正常姿勢(shì)和平衡。1分：左前爪能放在橫檔上，但當(dāng)該爪承重時(shí)滑下，同時(shí)大鼠掉落，影響正常行走。2分：左前爪能放在橫檔上，但當(dāng)該爪承重時(shí)滑下，大鼠未掉落，也不影響行走，能維持平衡和正常步態(tài)。3分：左前爪能放在橫檔上，但在承重前大鼠迅速抬起該爪并移到另一個(gè)橫檔上。4分：大鼠想將前爪放到一個(gè)橫檔上，但因沒(méi)接觸到該橫檔而放在另一橫檔上。5分：大鼠將左前爪放在橫檔上，但由腕關(guān)節(jié)、肘關(guān)節(jié)或爪趾承重。6分：大鼠能將左前爪正中部位放在橫檔上。

1.4.2 血腦屏障完整性檢測(cè)

術(shù)后48 h，每組各取5只大鼠，經(jīng)左股靜脈注入2%伊文思藍(lán)(Evans blue,EB)5 ml/kg。3 h后10%水合氯醛4 ml/kg腹腔注射麻醉大鼠，左心室灌注生理鹽水300 ml，斷頭取腦，將損傷側(cè)腦組織浸泡在甲酰胺800 μl中，60℃避光48 h。離心取上清液，用酶標(biāo)儀檢測(cè)(λ=632 nm)光密度值。每個(gè)樣本重復(fù)檢測(cè)3次。根據(jù)標(biāo)準(zhǔn)曲線計(jì)算大鼠腦組織中EB含量(μg/g)。

1.4.3 Western blotting

術(shù)后7 d，每組3只大鼠10%水合氯醛4 ml/kg腹腔注射麻醉，斷頭取腦，迅速于冰上分離大腦皮層，干冰速凍，-80℃冰箱保存。勻漿，冰上超聲裂解。4℃、14,000 g離心30 min，取上清?？捡R斯亮藍(lán)法測(cè)定蛋白濃度。配分離膠和積層膠，上樣量50 μg，60 V電泳至樣品進(jìn)入分離膠中，100 V電泳至溴酚藍(lán)至凝膠末端；轉(zhuǎn)膜電壓100 V，時(shí)間90 min，5%脫脂奶粉室溫封閉l h。加MMP-2抗體(1∶1500，ABCAM公司)，室溫振蕩1 h，4℃過(guò)夜。GAPDH(1∶1500，EPITOMICS公司)作為內(nèi)參。加HRP標(biāo)記的山羊抗兔IgG二抗(1∶4000，上海舒濟(jì)生物公司)，室溫振蕩l h。加熒光顯影劑，凝膠成像系統(tǒng)成像，Quantity One圖像分析系統(tǒng)(BIO-RAD公司)分析蛋白條帶光密度。將假手術(shù)組光密度設(shè)為1，計(jì)算運(yùn)動(dòng)組和對(duì)照組光密度與假手術(shù)組光密度的比值。

1.5 統(tǒng)計(jì)學(xué)分析

采用SPSS 20.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)以(xˉ±s)表示，足誤試驗(yàn)評(píng)分采用重復(fù)測(cè)量方差分析，血腦屏障和Western blotting數(shù)據(jù)采用獨(dú)立樣本t檢驗(yàn)。顯著性水平α=0.05。

2 結(jié)果

2.1 足誤試驗(yàn)

假手術(shù)組足誤評(píng)分5.5分左右。對(duì)照組術(shù)后6 d評(píng)分明顯降低，以后無(wú)明顯增高的趨勢(shì)，基本穩(wěn)定在4.2分左右。運(yùn)動(dòng)組術(shù)后6 d評(píng)分降低，12 d、18 d及24 d評(píng)分有所升高，且高于對(duì)照組(P＜0.05)。見(jiàn)表1。

表1 運(yùn)動(dòng)組與對(duì)照組各時(shí)間點(diǎn)足誤試驗(yàn)評(píng)分比較

2.2 血腦屏障完整性

假手術(shù)組基本未見(jiàn)藍(lán)染組織。對(duì)照組皮層和胼胝體明顯呈藍(lán)色，運(yùn)動(dòng)組皮層和胼胝體藍(lán)染組織減少。運(yùn)動(dòng)組EB滲出量(14.6±1.14)μg/g，顯著低于對(duì)照組(23.5±2.18)μg/g(t=-8.091,P＜0.001)。

2.3 Western blotting

對(duì)照組MMP-2蛋白相對(duì)表達(dá)量為(2.51±0.11)，顯著高于運(yùn)動(dòng)組(1.58±0.17)(t=-13.12,P＜0.001)。見(jiàn)圖1。

圖1 各組Western blotting結(jié)果

3 討論

血腦屏障主要由腦微血管內(nèi)皮細(xì)胞、星形膠質(zhì)細(xì)胞終足、基底膜和周細(xì)胞構(gòu)成。腦微血管內(nèi)皮細(xì)胞依靠緊密連接構(gòu)成一個(gè)連續(xù)密閉的屏障，不僅防止血液循環(huán)中的大分子物質(zhì)進(jìn)入腦實(shí)質(zhì)，也承擔(dān)必要的物質(zhì)交換，是維持神經(jīng)系統(tǒng)內(nèi)穩(wěn)定的重要成分。血腦屏障受損可增加血源性中性粒細(xì)胞和白細(xì)胞在損傷血管內(nèi)皮細(xì)胞處聚集和穿越至腦實(shí)質(zhì)，加劇腦實(shí)質(zhì)神經(jīng)炎性反應(yīng)，加重腦損傷[13]。

腦水腫是腦外傷急性期重要的病理過(guò)程，表現(xiàn)為腦組織含水量和腦體積增高。腦外傷引發(fā)腦水腫主要有兩種機(jī)制，即細(xì)胞毒性腦水腫和血管源性腦水腫。MMPs是一組Zn2+依賴的可降解或修飾細(xì)胞外基質(zhì)的蛋白酶，生理狀態(tài)下以酶原形式存在。細(xì)胞毒性水腫后細(xì)胞持續(xù)腫脹，引起細(xì)胞壞死，腦局部滲透狀態(tài)改變，激活MMPs；激活的MMPs可降解基底膜Ⅳ型膠原等成分，破壞血腦屏障完整性，從而引起毛細(xì)血管通透性增加，導(dǎo)致血漿成分和大分子物質(zhì)外滲，形成血管源性腦水腫和出血轉(zhuǎn)化，進(jìn)一步惡化腦水腫[14-16]。

許多腦外傷動(dòng)物模型顯示，MMP-2和MMP-9在腦外傷后數(shù)小時(shí)至數(shù)天表達(dá)顯著增加[17-19]。臨床上中重度腦外傷患者血漿和腦脊液可檢測(cè)到MMP-2和MMP-9升高[20-21]。MMP-2和MMP-9的高表達(dá)與ICU住院時(shí)間延長(zhǎng)和死亡率增高密切相關(guān)[22-23]。目前普遍認(rèn)為，MMPs表達(dá)上調(diào)和活性增強(qiáng)是早期腦損傷的重要因素之一[24]。

本研究顯示，早期運(yùn)動(dòng)訓(xùn)練能改善腦外傷大鼠運(yùn)動(dòng)協(xié)調(diào)功能，與以前的報(bào)道一致[9-12]。本研究也發(fā)現(xiàn)，腦外傷后大鼠血腦屏障破壞[25]，通透性增加；而運(yùn)動(dòng)訓(xùn)練能減輕腦外傷后血腦屏障的破壞，一定程度上維持血腦屏障的完整性。這可能與抑制腦外傷后MMP-2蛋白表達(dá)增加有關(guān)。

由于神經(jīng)炎癥和免疫與血腦屏障的密切聯(lián)系，在這些方面展開(kāi)研究可能為進(jìn)一步闡明腦外傷后康復(fù)的作用機(jī)制提供依據(jù)。

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Effect of Treadmill Exercise on Expression of Matrix Metalloproteinase-2 in Rats with Traumatic Brain Injury

SHEN Xia-feng1,TIAN Shan2,WU Yi2,ZHU Yu-lian2
1.Department of Rehabilitation Medicine,The First Rehabilitaion Hospital of Shanghai,Shanghai 200092,China; 2.Department of Rehabilitation,Huashan Hospital,Fudan University,Shanghai 200040,China

ZHU Yu-lian.E-mail:hsyykfkzyl@163.com

Objective To determine the effect of treadmill exercise on expression of matrix metalloproteinase-2(MMP-2)in rats following traumatic brain injury(TBI).Methods Fifty-five male adult Sprague-Dawley rats were randomly assigned to sham(n=15),control(n= 20)and exercise(n=20)groups,the later two groups subjected to unilateral cortical contusion injury(CCI).All the rats were assessed with foot-fault test 6,12,18 and 24 days after CCI.Evans blue perfusion was used to evaluate the integrity of the blood-brain-barrier(BBB)48 hours after CCI.Protein expression of MMP-2 was determined with Western blotting one week after CCI.Results The score of foot-fault test improved more in the exercise group than in the control group 12,18 and 24 days after CCI(F＞4.793,P＜0.05).Evans blue extravasation was less in the exercise group than in the control group(t=-8.091,P＜0.001),as well as the expression of MMP-2(t=-13.12,P＜0.001). Conclusion Early treadmill exercise can improve the motor function in rats with TBI,which may associate with inhibition of MMP-2 expression to protect BBB integrity.

traumatic brain injury;treadmill exercise;blood-brain-barrier;matrix metalloproteinase;rats

R651.1

A

1006-9771(2017)05-0525-04

2017-03-08

2017-04-11)

10.3969/j.issn.1006-9771.2017.05.007

[本文著錄格式]沈夏鋒，田閃，吳毅，等.跑臺(tái)運(yùn)動(dòng)對(duì)腦外傷大鼠基質(zhì)金屬蛋白酶-2表達(dá)的影響[J].中國(guó)康復(fù)理論與實(shí)踐, 2017,23(5):525-528.

CITED AS:Shen XF,Tian S,Wu Y,et al.Effect of treadmill exercise on expression of matrix metalloproteinase-2 in rats with traumatic brain injury[J].Zhongguo Kangfu Lilun Yu Shijian,2017,23(5):525-528.

楊浦區(qū)科學(xué)技術(shù)委員會(huì)、衛(wèi)生和計(jì)劃生育委員會(huì)課題(No.YP15M13)。

1.上海市第一康復(fù)醫(yī)院康復(fù)科，上海市200092；2.復(fù)旦大學(xué)附屬華山醫(yī)院康復(fù)醫(yī)學(xué)科，上海市200040。作者簡(jiǎn)介：沈夏鋒(1970-)，男，漢族，浙江蒼南縣人，副主任醫(yī)師，主要研究方向：神經(jīng)康復(fù)。通訊作者：朱玉連。E-mail:hsyykfkzyl@163.com。