周　榮　丁小麗　劉良明

liangmingliu@yahoo.com

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失血性休克不同時(shí)相sorcin轉(zhuǎn)位及其與血管反應(yīng)性變化的相關(guān)性分析*

周榮丁小麗劉良明#

liangmingliu@yahoo.com

【摘要】目的：觀察失血性休克后不同時(shí)相大鼠腸系膜動(dòng)脈平滑肌sorcin蛋白表達(dá)及其對(duì)去甲腎上腺素(NE)的反應(yīng)性，初步探討sorcin是否參與失血性休克血管反應(yīng)性的調(diào)節(jié)。方法：36只SD大鼠，12只用作非手術(shù)對(duì)照組，其余采用經(jīng)股動(dòng)脈放血法制成失血性休克(動(dòng)脈壓40 mmHg)模型。分別將成模大鼠再分為休克早期(30min)組及晚期(2h)組；各組開(kāi)腹分離腸系膜動(dòng)脈，采用離體器官?gòu)埩y(cè)定技術(shù)檢測(cè)腸系膜動(dòng)脈對(duì)NE的反應(yīng)性；采用Western Blotting(WB)檢測(cè)腸系膜動(dòng)脈平滑肌sorcin的表達(dá)及其分布；采用免疫共沉淀聯(lián)合WB檢測(cè)sorcin-肌漿網(wǎng)2型雷諾定受體(RyR2)表達(dá)，并分析休克大鼠sorcin水平與腸系膜動(dòng)脈對(duì)NE反應(yīng)的相關(guān)性。結(jié)果：失血性休克早期組大鼠腸系膜動(dòng)脈對(duì)NE的收縮反應(yīng)較對(duì)照組明顯增強(qiáng)(P<0.05)，而晚期組對(duì)NE較對(duì)照組明顯降低(P<0.05)。早期組和晚期組大鼠腸系膜動(dòng)脈血管平滑肌sorcin總蛋白表達(dá)無(wú)顯著差異(P>0.05)，但發(fā)生了明顯轉(zhuǎn)位現(xiàn)象，即早期組sorcin在胞漿及質(zhì)膜內(nèi)表達(dá)無(wú)明顯差異(P>0.05)，但晚期組sorcin在胞漿內(nèi)的表達(dá)明顯增強(qiáng)，而在質(zhì)膜的表達(dá)顯著降低(均P<0.05)；免疫共沉淀發(fā)現(xiàn)，早期組大鼠腸系膜動(dòng)脈平滑肌sorcin-RyR2復(fù)合子表達(dá)與對(duì)照組無(wú)顯著性差異(P>0.05)，而晚期組較對(duì)照組明顯減少(P<0.05)；相關(guān)性分析顯示，晚期組sorcin由質(zhì)膜向胞漿移位與其血管反應(yīng)性呈顯著負(fù)相關(guān)(P<0.01)。結(jié)論：失血性休克后2 h，大鼠腸系膜動(dòng)脈平滑肌sorcin從RyR2解離，導(dǎo)致sorcin從肌漿網(wǎng)向胞漿轉(zhuǎn)位，至少部分參與腸系膜動(dòng)脈對(duì)NE低反應(yīng)性的形成。

【關(guān)鍵詞】失血性休克；血管反應(yīng)性；sorcin蛋白；肌漿網(wǎng)2型雷諾定受體；大鼠

嚴(yán)重創(chuàng)傷失血常引起外周血管對(duì)血管活性物質(zhì)如去甲腎上腺素(NE)、精氨酸加壓素等的反應(yīng)性異常[1]。我們的前期工作證實(shí)，血管平滑肌肌漿網(wǎng)2型雷諾定受體(Type Ⅱ Ryanodine Receptor, RyR2)的過(guò)度激活與失血性休克晚期血管低反應(yīng)性形成有關(guān)[2，3]，但其通過(guò)何種機(jī)制導(dǎo)致RyR2的過(guò)度激活目前尚未闡明。

基礎(chǔ)研究表明， sorcin是參與調(diào)節(jié)肌漿網(wǎng)RyR2介導(dǎo)Ca2+釋放通道活性的重要蛋白分子，其通過(guò)與RyR2胞漿段sorcin相應(yīng)位點(diǎn)結(jié)合，抑制RyR2活性及其介導(dǎo)的肌漿網(wǎng)Ca2+釋放[4]；sorcin與RyR2間親和力的減弱可能引起血管平滑肌膜超極化及血管舒張，如F112L-sorcin突變可導(dǎo)致sorcin與RyR2間親和力減弱6倍，觸發(fā)血管平滑肌細(xì)胞(Vascular Smooth Muscle Cell，VSMC)鈣火花及瞬時(shí)外向鉀電流(STOCs)的形成[4，5]。 STOCs通過(guò)激活大電導(dǎo)鈣激活鉀通道(Large Conductance Calcium Activated Potassium Channel，BKCa)，促成休克晚期血管低反應(yīng)性的形成[6, 7]。由此推測(cè)，失血性休克時(shí)外周血管可能發(fā)生sorcin與RyR2解離，并可能與休克晚期血管低反應(yīng)性的發(fā)生有關(guān)。

本研究通過(guò)建立大鼠失血性休克模型，觀察失血性休克不同時(shí)期sorcin在VSMC胞漿及質(zhì)膜的表達(dá)變化、sorcin-RyR2復(fù)合子形成和解離情況及其與休克血管異常反應(yīng)性之間的關(guān)系，為尋求休克時(shí)血管低反應(yīng)性恢復(fù)劑提供實(shí)驗(yàn)依據(jù)。

1材料與方法

1.1實(shí)驗(yàn)動(dòng)物和試劑

SD大鼠(體重200±20g，雌雄不限)由第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所實(shí)驗(yàn)動(dòng)物中心提供(動(dòng)物合格證號(hào)：SYXK2002-032)；NE購(gòu)自遠(yuǎn)大醫(yī)藥(中國(guó))有限公司，批號(hào)140507(2 mg/mL)，采用0.01mol/L HCl配制；戊巴比妥鈉、抗β-actin單抗購(gòu)自Sigma-Aldrich公司(美國(guó))；Supersignal發(fā)光增強(qiáng)試劑盒購(gòu)自Pierce公司(美國(guó))；Protein G、抗RyR2抗體、抗sorcin抗體購(gòu)自Santa Cruz公司(美國(guó))。其它相關(guān)試劑為國(guó)產(chǎn)分析純。

1.2分組及失血性休克模型

采用隨機(jī)數(shù)字表法將實(shí)驗(yàn)大鼠分為對(duì)照組(n=12)和模型組(n=24)。模型組參照文獻(xiàn)[8]建立失血性休克大鼠在體模型：實(shí)驗(yàn)前，大鼠禁食12h，自由飲水，建模當(dāng)日戊巴比妥鈉(30mg/kg)腹腔注射麻醉后，右側(cè)股動(dòng)脈插管并接血壓計(jì)，肝素鈉500U/kg抗凝，術(shù)畢血壓穩(wěn)定10min后開(kāi)始放血，在10min內(nèi)將平均動(dòng)脈壓(MAP)降至40mmHg為失血性休克成模，并在此水平分別維持30min(n=12，休克早期組)和2h(n=12，休克晚期組)。對(duì)照組不行手術(shù)，普通飼養(yǎng)。

1.3觀察指標(biāo)及其檢測(cè)方法

1.3.1離體血管張力：各組大鼠開(kāi)腹取出整個(gè)腸組織，在解剖顯微鏡下剝離腸系膜上動(dòng)脈主干及其1-2級(jí)分支，并清除周圍結(jié)締組織。動(dòng)脈主干切取1-2個(gè)血管環(huán)，余下組織用于蛋白提取(見(jiàn)后述)。將血管環(huán)置于Krebs-Henseleit(K-H)液(mmol/L：NaCl 118、 KCl 4.7、 NaHCO325、 KH2PO41.03、

MgSO4·7H2O 0.45、CaCl22.5、EDTA 0.001、glucose 11.1，pH 7.4)。在解剖顯微鏡下將血管環(huán)懸掛于一對(duì)不銹鋼絲上，一端置于固定柱，另一端與張力換能器(PowerLab，澳大利亞)相連，浸于注有K-H液的離體器官灌流浴槽中(37℃恒溫)孵育，持續(xù)通氧。待血管張力平衡后，記錄NE誘導(dǎo)血管環(huán)收縮曲線。血管環(huán)對(duì)NE(10-9-10-5mol/L)的收縮反應(yīng)采用濃度累計(jì)法測(cè)定，即依次加入終濃度為10-9、10-8、10-7、10-6、10-5mol/L的NE，記錄早、晚休克組不同NE濃度下血管環(huán)收縮反應(yīng)的變化，以加NE后血管環(huán)的收縮力與血管環(huán)初始張力的差值作量-效曲線，以量-效曲線及10-5mol/L NE誘導(dǎo)的最大收縮力(Emax，g)評(píng)價(jià)血管對(duì)NE的收縮反應(yīng)性[1]。

1.3.2胞漿及質(zhì)膜蛋白的提?。簠⒄毡緦?shí)驗(yàn)室方法[9]進(jìn)行血管平滑肌胞漿及質(zhì)膜蛋白的提取。將去內(nèi)皮血管組織置于預(yù)冷蛋白裂解液中(mM: Tris-HCl 20、EDTA 2、EGTA 1、β-巰基乙醇 0.1%、NaF 10、Na3VO41、protein kinase inhibitor colktail pit)，于冰浴中剪碎組織并勻漿，收集上清置冰浴中凍融1h；上清經(jīng)1 000g離心10min(4℃)，取上清再16 000g離心30 min (4℃)，收集上清即為胞漿總蛋白；將沉淀用質(zhì)膜蛋白裂解液(mM: Tris-HCl 20、EDTA 2、EGTA 1、β-巰基乙醇 0.1%、NaF 10、Na3VO41、TritonX 100 1%、SDS 0.1%)溶解后，經(jīng)16 000g離心20 min(4℃)，收集上清即為質(zhì)膜總蛋白。采用Brad-ford法進(jìn)行蛋白定量。

1.3.3胞漿和質(zhì)膜sorcin蛋白表達(dá)： 采用Western blotting(WB)檢測(cè)各組大鼠血管平滑肌sorcin的表達(dá)。參照文獻(xiàn)[10]，取等量樣品蛋白(120μg/道)經(jīng)15% SDS-PAGE凝膠電泳分離后轉(zhuǎn)移至硝酸纖維素膜，轉(zhuǎn)移完畢后用5% 脫脂奶粉室溫封閉4 h后加入抗sorcin單抗(1∶800)室溫孵育4 h，再加入辣根酶標(biāo)記二抗(1∶10 000)室溫孵育1h，將待測(cè)硝酸纖維素膜置于supersignal發(fā)光增強(qiáng)試劑中反應(yīng)2min-3min后，迅速用塑料膜包裹硝酸纖維素膜，與X-ray膠片一同放入暗盒中曝光2min-5min，再將膠片顯影、定影，確定蛋白條帶的相應(yīng)分子量。將膠片置于圖像分析儀(Bio-Rad Gel 2000型,美國(guó))中，對(duì)目標(biāo)條帶進(jìn)行掃描及灰度分析。

1.3.4Sorcin-RyR2復(fù)合子檢測(cè)： 采用免疫共沉淀聯(lián)合免疫雜交檢測(cè)Sorcin-RyR2復(fù)合子的形成及解離情況。收集各組總蛋白(500μl)，加入抗RyR2單抗(5μg)，4℃振搖(over-night)，繼續(xù)加入protein G (20 μl) 4℃振搖5 h后，2 500 g離心10 min(4℃)，棄上清；采用RIPA洗滌3次后，加入RIPA和上樣緩沖液(4∶1)，100℃煮5 min。常規(guī)SDS-PAGE電泳后轉(zhuǎn)移至硝酸纖維素膜，加入抗sorcin單抗(1∶1 000)，室溫孵育4 h，再加入辣根酶標(biāo)記二抗(1∶10 000)，室溫孵育1 h，將待測(cè)硝酸纖維素膜置于supersignal發(fā)光增強(qiáng)試劑中反應(yīng)2min-3min后，迅速用塑料膜包裹硝酸纖維素膜，與X-ray膠片一同放入暗盒中曝光2min-5min，再將膠片顯影、定影，確定蛋白條帶的相應(yīng)分子量。將膠片置于圖像分析儀中，對(duì)目標(biāo)條帶進(jìn)行掃描及灰度分析。

1.4統(tǒng)計(jì)學(xué)處理

2結(jié)果

2.1各組大鼠腸系膜動(dòng)脈對(duì)NE的反應(yīng)性

結(jié)果顯示，休克早期組大鼠腸系膜動(dòng)脈對(duì)NE的收縮反應(yīng)性顯著高于對(duì)照組，表現(xiàn)為NE的量-效曲線明顯左移，10-5mol/L NE引起血管Emax由0.72±0.15g升至1.11±0.28g(t=1.931，P<0.05)；休克晚期組腸系膜動(dòng)脈對(duì)NE的收縮反應(yīng)性較對(duì)照組明顯降低，表現(xiàn)為NE的量-效曲線明顯右移，10-5mol/L NE引起血管Emax由0.72±0.15g降至0.50±0.11g(t=1.874，P<0.05)，與我們以往的報(bào)道[1]一致。見(jiàn)圖1。

注：與對(duì)照組比較，*P<0.05

2.2各組大鼠腸系膜動(dòng)脈平滑肌sorcin在胞漿及質(zhì)膜的表達(dá)

結(jié)果顯示，各組sorcin總表達(dá)量差異無(wú)統(tǒng)計(jì)學(xué)意義(F=5.607，P>0.05)；失血性休克早期組，sorcin在血管平滑肌胞漿及質(zhì)膜中的表達(dá)無(wú)顯著差異(t=1.131，P>0.05)，而晚期組胞漿sorcin表達(dá)升高，質(zhì)膜sorcin表達(dá)降低，兩者差異有統(tǒng)計(jì)學(xué)意義(t=1.868，P<0.05)。提示失血性休克晚期，sorcin可從肌漿網(wǎng)向胞漿轉(zhuǎn)位。見(jiàn)圖2。

注：與休克早期組比較，*P<0.01

2.3休克早期組和晚期組sorcin-RyR2復(fù)合子表達(dá)

結(jié)果顯示，失血性休克早期組與對(duì)照組大鼠腸系膜動(dòng)脈平滑肌sorcin-RyR2復(fù)合子表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(t=1.319，P>0.05)，晚期組sorcin-RyR2復(fù)合子表達(dá)較對(duì)照組顯著減少，差異有統(tǒng)計(jì)學(xué)意義(t= 2.067，P<0.05)，見(jiàn)圖3。

2.4休克晚期組大鼠Sorcin胞漿水平與腸系膜動(dòng)脈對(duì)NE反應(yīng)性的相關(guān)性分析

相關(guān)分析結(jié)果顯示，休克晚期組大鼠sorcin在胞漿的表達(dá)與腸系膜動(dòng)脈對(duì)NE反應(yīng)性(收縮力)呈顯著負(fù)相關(guān)(r=-0.983,P<0.01)；休克早期組大鼠sorcin在胞漿的表達(dá)與腸系膜動(dòng)脈對(duì)NE反應(yīng)性未見(jiàn)顯著相關(guān)性(r=0.087,P>0.05)。見(jiàn)圖4。

注：與對(duì)照組比較，*P<0.05

圖4　失血性休克晚期組大鼠腸系膜動(dòng)脈平滑肌胞漿sorcin

3討論

嚴(yán)重創(chuàng)傷/失血性休克后血管功能障礙的發(fā)生是導(dǎo)致臨床患者血壓持續(xù)下降、器官灌流不足及機(jī)體死亡的重要原因之一。肌漿網(wǎng)由RyR介導(dǎo)的內(nèi)鈣釋放是調(diào)控血管平滑肌舒-縮反應(yīng)的重要機(jī)制之一，其在失血性休克血管反應(yīng)性異常發(fā)生中的作用研究甚少。

分布于血管平滑肌的sorcin調(diào)節(jié)著血管平滑肌細(xì)胞興奮-收縮偶聯(lián)。生理狀態(tài)下，位于胞漿的sorcin選擇性結(jié)合RyR2并維持RyR2/鈣釋放通道處于閉合狀態(tài)，從而抑制RyR2的基礎(chǔ)活性[11]。本研究結(jié)果顯示，在失血性休克大鼠腸系膜動(dòng)脈去內(nèi)皮后，sorcin的表達(dá)發(fā)生明顯轉(zhuǎn)位，即在失血性休克晚期(2h)，sorcin在胞漿的表達(dá)明顯升高，而在質(zhì)膜的表達(dá)顯著降低；同時(shí)發(fā)現(xiàn)，失血性休克晚期大鼠腸系膜動(dòng)脈血管平滑肌sorcin-RyR2復(fù)合子表達(dá)水平較對(duì)照組顯著降低。表明在失血性休克晚期發(fā)生了sorcin與RyR2解離及從肌漿網(wǎng)向胞漿轉(zhuǎn)位；而且sorcin在胞漿中的表達(dá)升高與血管反應(yīng)性降低呈顯著負(fù)相關(guān)。而在失血性休克早期(30min)，sorcin在胞漿及質(zhì)膜的表達(dá)、sorcin-RyR2復(fù)合子水平以及血管對(duì)NE的收縮反應(yīng)性均與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義。

已有研究顯示，sorcin從RyR2的解離可刺激RyR2通道活性及RyR2介導(dǎo)肌漿網(wǎng)內(nèi)鈣釋放的增加，通過(guò)觸發(fā)鈣火花及STOCs形成[4，5]，導(dǎo)致VSMC膜BKCa通道的激活[6]；我們以往的研究也表明，BKCa通道的過(guò)度激活促成了休克晚期血管低反應(yīng)性的形成。因而認(rèn)為sorcin從RyR2解離可能與失血性休克后血管低反應(yīng)性的形成有關(guān)。至于sorcin從RyR2解離在觸發(fā)休克血管低反應(yīng)性形成中的可能機(jī)制，以及休克晚期觸發(fā)sorcin與RyR2解離的上游調(diào)控分子，均有待進(jìn)一步探討。

綜上，失血性休克晚期，大鼠腸系膜動(dòng)脈sorcin與肌漿網(wǎng)RyR2解離所導(dǎo)致的sorcin從肌漿網(wǎng)質(zhì)膜向胞漿轉(zhuǎn)位，可能是促成失血性休克晚期血管低反應(yīng)性形成的重要原因。

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周榮(1971-)，女，漢族，醫(yī)學(xué)博士，副研究員，研究方向?yàn)樾菘搜芄δ苷系K發(fā)生機(jī)制及防治

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2Zhou R, Chen F, Li Q, et al. Stimulation of the adenosine A3receptor reverses vascular hyporeactivity hyporeactivity after hemorrhagic shock in rats[J]. Acta Pharmacol Sin,2010,31(4):413-420.

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4Franceschini S, Ilari A, Verzili D, et al. Molecular basis for the impaired function of the natural F112L sorcin mutant: X-ray crystal structure, calcium affinity, and interaction with annexin VII and the ryanodine receptor[J]. FASEB J,2008,22(1):295-306.

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The Translocation of Sorcin and the Correlating Analysis with the Changes of Vascular Reactivity after Hemorrhagic Shock

ZHOU Rong，DING Xiao-li，LIU Liang-ming#

State Key Laboratory of Trauma, Burns and Combained Injury, Second Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China；#Corresponding author

【Abstract】Objective: To explore the expression and distribution of sorcin in superior mesenteric artery (SMA) during different stage after hemorrhagic shock and its role in the regulation of vascular reactivity after hemorrhagic shock. Method: 30 SD rats were divided into two groups, twelve as a nonsurgical control group and the rest of the rats were classified as hemorrhagic shock model group that suffered from femoral artery bleeding method (arterial pressure is 40mmHg). The model group rats were divided into the early stage of shock group (30min) and the late stage of shock group (2h). Isolated mesenteric arteries were isolated from each group. The reactivity of mesenteric artery to NE was detected by using in vitro organ tension test. The expression and distribution of sorcin protein in mesenteric artery smooth muscle was detected by using Western Blotting (WB). The expression of sorcin-RyR2 was detected by using Co-immunoprecipitation combined with WB. Then analyze the correlation between the level of sorcin protein and the reactivity of mesenteric artery to NE in the shock rats.Results: During the early stage (30min) after hemorrhagic shock, the vascular reactivity to NE increased significantly, while the vascular reactivity to NE decreased during late stage (2h), which suggested that there was bi-phasic vascular reactivity to NE in SMA during different stage after hemorrhagic shock. The total expression of sorcin had no significant changes but the distribution of sorcin in cytoplasm and plasmalemma fraction of VSMC changed significantly, characterized with the expression of sorcin in cytoplasm and in plasmalemma had no significant changes during the early stage (30min) after hemorrhagic shock, while the expression of sorcin in cytoplasm fraction was upregulated but its expression in plasmalemma fraction decreased significantly during the late stage (2h) after hemorrhagic shock. Immunoprecipitation analysis showed that formation of sorcin-RyR2 complex decreased during the late stage but not during the early stage after hemorrhagic shock. The correlation analysis showed that the increased expression of sorcin in cytoplasm closely associated with the blunted vascular hyporeactivity to NE during the late stage after hemorrhagic shock. Conclusion: At late stage after hemorrhagic shock, sorcin translocated from cytoplasm to plasmalemma in superior mesenteric artery in rat. The dissociation of sorcin from RyR2 at least partly associated with the development of vascular hyporeactivity to NE during the late stage after hemorrhagic shock.

【Key words】Hemorrhagic shock; Vascular reactivity; Sorcin; Ryanodine receptor Ⅱ; Rat

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作者簡(jiǎn)介：本文第一

[中圖分類號(hào)]R541.6+4

[文獻(xiàn)標(biāo)識(shí)碼]A

[文章編號(hào)]1005-1740(2016)01-0001-05

*[基金項(xiàng)目]國(guó)家自然科學(xué)基金(81100227、81370427)

本文2015-07-09收到，2015-12-20修回