李煥敏，李春光

(1. 南方醫(yī)科大學(xué)第三附屬醫(yī)院神經(jīng)內(nèi)科，廣州 510630； 2.南方醫(yī)科大學(xué)珠江醫(yī)院神經(jīng)內(nèi)科，廣州 510280)

研究報告

基質(zhì)金屬蛋白酶9及其組織型抑制因子1在高血壓大鼠血液及組織中的變化

李煥敏1，李春光2*

(1. 南方醫(yī)科大學(xué)第三附屬醫(yī)院神經(jīng)內(nèi)科，廣州 510630； 2.南方醫(yī)科大學(xué)珠江醫(yī)院神經(jīng)內(nèi)科，廣州 510280)

目的 探討腎血管性高血壓大鼠(RHR) 血清、腦及血管組織中基質(zhì)金屬蛋白酶9(MMP-9) 和組織型基質(zhì)金屬蛋白酶抑制因子1(TIMP-1)的表達以及血壓與兩者的關(guān)系。方法 雄性SD大鼠80只隨機分成RHR組(40只) 和假手術(shù)組(40只)。RHR組采用雙腎-雙夾法制作高血壓大鼠模型，鼠尾測壓儀測定血壓。根據(jù)Longa 5評分法和病理學(xué)結(jié)果確定腦卒中；Western blot和免疫組化測定腦組織和血管中MMP-9和TIMP-1的表達，雙抗體夾心ELISA 檢測血清MMP-9和TIMP-1水平。結(jié)果 與假手術(shù)組比較，RHR組大鼠術(shù)后2、4、6、8、10、12 周血壓均明顯升高[(157±9.0)比(128±7.0)， (176±10.0)比(122±6.0)， (194±8.0)比(117±6.5)， (202±12.0)比(124±8.0)， (218±15.0)比(126.±8.5)， (224±20.0)比(129.±9.0) mmHg，均P< 0.05]；術(shù)后12周，RHR大鼠血清MMP-9高于假手術(shù)組[(783.4±109.79)比(573.4±109.59) ng/mL，P<0.05]；而RHR大鼠血清TIMP-1低于假手術(shù)組[(313.02±83.9)比(976.19±191.1) pg/mL，P<0.05]。同時，RHR大鼠腦組織和血管MMP-9的表達均明顯高于假手術(shù)組 (均P<0.05)，而TIMP-1的表達則均明顯低于假手術(shù)組(均P<0.05)。Pearson相關(guān)分析顯示收縮壓與血清、腦及血管組織中的MMP- 9水平均呈正相關(guān)(r=0.557，r=0.774和r=0.661，均P<0.05)，而與TIMP-1水平均呈負相關(guān)(r=-0.481，r=-0.535和r=-0.685，均P<0.01)。結(jié)論 腎血管性高血壓大鼠血清、腦和血管組織中MMP-9表達升高，而TIMP-1表達降低。兩者在血液和組織中的變化趨勢保持一致，血壓的升高與血液和組織中MMP-9的升高和TIMP-1的降低相關(guān)。

腎血管性高血壓大鼠；基質(zhì)金屬蛋白酶9；組織型基質(zhì)金屬蛋白酶抑制因子1

高血壓是心腦血管病的主要危險因素，基質(zhì)金屬蛋白酶-9(matrix metalloproteinase-9，MMP-9)在高血壓相關(guān)的心腦血管疾病的病理生理過程中發(fā)揮重要作用[1-3]。循環(huán)中MMP-9水平可預(yù)測冠心病和腦卒中患者的心血管疾病死亡率[4, 5]。在無心血管疾病的人群中，升高的MMP-9與急性心血管事件和/或高血壓相關(guān)[6]。因此，循環(huán)中MMP-9的升高可能使健康人群更易罹患心腦血管疾病。TIMP-1(tissue inhibitor of metalloproteinase-1，TIMP-1)是MMP-9 主要的內(nèi)源性抑制劑[7]，TIMP-1 通過與MMP-9 的鋅離子結(jié)合而覆蓋在其活性中心部位，通過空間位阻效應(yīng)使底物不能與活性中心結(jié)合從而發(fā)揮抑制作用。在生理狀態(tài)下，兩者在體內(nèi)同步表達，以1∶1 的形式構(gòu)成復(fù)合體。在病理條件下這種平衡被打破會導(dǎo)致相應(yīng)的病理損害[8]。本研究擬通過制作腎血管性高血壓大鼠(renovascular hypertensive rats，RHR) 模型，觀察RHR模型血清、腦組織及血管中MMP-9和TIMP-1的變化，探討高血壓對MMP-9和TIMP-1的影響。

1 材料與方法

1.1 實驗動物

選用SPF級雄性SD大鼠80 只，體重80～120 g，鼠齡2月齡，將大鼠隨機分成RHR組40只和假手術(shù)組40只。均購于廣東省醫(yī)學(xué)實驗動物中心【SCXK(粵)2013-0002】，以普通顆粒性大鼠飼料(蛋白質(zhì)23%、脂肪4.7%、鈉鹽0.24%)喂養(yǎng)，飲用自來水，12 h循環(huán)燈光，恒定濕度，室溫(23±3)℃，實驗大鼠飼養(yǎng)及組織取材均于中山大學(xué)實驗動物中心實驗設(shè)備內(nèi)進行【SYXK(粵)2012-0081】。

1.2 方法

1.2.1 高血壓模型大鼠制作

RHR組大鼠按雙腎-雙夾法制作。具體方法如下：手術(shù)前12 h禁食，用10%水合氯醛(250 mg/kg)腹腔注射麻醉，仰臥固定，常規(guī)消毒鋪巾，經(jīng)腹部正中縱行切口，依次分離雙側(cè)腎動脈，用自制“Ω”環(huán)形銀夾(內(nèi)徑為0.2 mm)分別鉗夾雙側(cè)腎動脈起始部，使腎動脈置于銀夾的環(huán)形結(jié)構(gòu)中，夾子能夠沿動脈滑動，并確認雙腎無明顯的瘀血、壞死或蒼白，整個手術(shù)過程不損傷腎、肝、乳糜池及腎靜脈。對腹膜和肌肉用連續(xù)縫合，皮膚用間斷縫合。術(shù)后腹腔注射慶大霉素素(8000 U/kg)預(yù)防感染。假手術(shù)組大鼠只開腹分離腎動脈，不放置銀夾，其余步驟同前。術(shù)后4～6 h恢復(fù)喂食，術(shù)后注意保暖，每天早晚觀察大鼠肢體活動及呼吸、進食、傷口愈合情況。在飼養(yǎng)過程中出現(xiàn)腦卒中癥狀及體征的大鼠均被剔除并進行更換。術(shù)后4 周收縮壓>150 mmHg 為造模成功。假手術(shù)組僅打開腹腔，不上銀夾，其他步驟同RHR組。兩組術(shù)后均用8000 U/ kg 慶大霉素腹腔內(nèi)注射預(yù)防感染。術(shù)后4～6 h恢復(fù)喂食，術(shù)后注意保暖，每天早晚觀察大鼠肢體活動及呼吸、進食、傷口愈合情況。

1.2.2 血壓測定

將大鼠于37℃溫箱中預(yù)熱約15 min，預(yù)熱后采用BP-98A型Softron大鼠心率血壓計經(jīng)尾動脈測量血壓。每只大鼠測3次，取平均值作為該大鼠的血壓值。術(shù)前3 d連續(xù)測血壓作為基礎(chǔ)血壓，術(shù)后2、4、6、8、10、12 周固定時間點各測1次。

1.2.3 標(biāo)本制作

大鼠腹腔麻醉后經(jīng)升主動脈快速灌注肝素化生理鹽水(50 U/mL) 120 mL (滴速為10 mL/min) 以清除血管內(nèi)血液，斷頭取腦，分離主動脈，腦組織在冰箱-20℃冷凍10 min后由嘴側(cè)至尾側(cè)每2 mm切成一冠狀薄片，先肉眼觀察出血情況，再TTC染色觀察梗死情況。每亞組隨機取8只大鼠進行Western blot檢測：切取主動脈及部分額葉，快速置入液氮中，成固體狀后放-80℃冰箱保存。將其余大鼠腦及主動脈組織于4%多聚甲醛溶液中固定，常規(guī)脫水，石蠟包埋，連續(xù)切片進行免疫組化和HE染色，鏡檢觀察。

1.2.4 Western blot

樣品制備，BCA法測量蛋白濃度，配膠(5%濃縮膠，12%分離膠)，上樣(20 μg)。①電泳：100V恒壓15 min，150 V恒壓電泳至溴酚藍剛出膠底部止。②轉(zhuǎn)膜：制作電轉(zhuǎn)“三明治”恒壓100 V， MMP-9(90 min)，TIMP-1(30 min)。內(nèi)參：GAPDH(40 min)。封閉：轉(zhuǎn)膜結(jié)束后，將膜取下，用TBST漂洗3次，每次5 min，根據(jù)目的蛋白和GAPDH蛋白大小將膜切開，將膜置于封閉液中；TBST洗膜；結(jié)合一抗：分別將MMP-9(購自Cell Signaling Technology公司)和TIMP-1抗體(購自Santa Cruz公司)用5%脫脂奶粉封閉液按1∶750稀釋后、GAPDH抗體按1∶10 000稀釋后，將PVDF膜放置其中，4℃過夜孵育；TBST洗膜5 min，三次。結(jié)合二抗：將膜轉(zhuǎn)移到含有辣根過氧化酶(HRP)標(biāo)記的羊抗兔多克隆抗體的新鮮封閉液中，室溫下放置在脫色搖床上，室溫平緩搖動40 min；TBST洗膜5 min，三次。蒸餾水漂洗膜2 min，棄去液體。共洗三次。將雜交膜置于一透明塑料板上，注意不要讓膜干燥。用一干凈移液器將化學(xué)熒光發(fā)光底物均勻地加到膜的表面，并使反應(yīng)持續(xù)5 min。用試劑盒提供的濾紙吸去膜表面多余的底物溶液，放至暗盒，顯影。

1.2.5 免疫組化

每組隨機選取8只大鼠，將大鼠腦和主動脈經(jīng)過石蠟包埋后在切片機上連續(xù)切片作MMP-9和TIMP-1免疫組化染色。具體步驟如下：用二甲苯脫蠟，梯度酒精水化，3% H2O2封閉內(nèi)源性過氧化物酶，微波加熱抗原修復(fù)，10% 正常羊血清孵育，滴加兔抗鼠MMP-9(購自Cell Signaling Technology公司)或TIMP-1一抗(1∶100)(購自Santa Cruz公司)，37℃孵育50 min，0.01 mol/L PBS漂洗，滴加生物素化羊抗兔二抗(購自Dako公司)，37℃孵育40 min，0.01 mol/L PBS漂洗，DAB 顯色5～10 min，蒸餾水漂洗，蘇木精復(fù)染，梯度酒精脫水，二甲苯透明，中性樹脂封片觀察。免疫組化陽性細胞胞質(zhì)或胞核內(nèi)可見棕黃色顆?；驁F塊狀物質(zhì)，在400 倍光鏡下隨機選取腦皮層和主動脈的20 個互不重疊的視野拍照，所選區(qū)域占整個動脈環(huán)和腦皮層的20%～30%，并且使批間變異小于4%。利用Image-Pro plus 6.0軟件計算平均光密度。

1.2.6 血清MMP-9和TIMP-1水平測定

術(shù)后12周，將大鼠用10%水合氯醛(300 mg/kg) 腹腔注射麻醉后開腹經(jīng)下腔靜脈采血，EDTA-K2抗凝，離心20 min(3000 r/min)，收集上清，采用ELISA法(購自上海西唐生物科技有限公司) 測定血清MMP-9和TIMP-1水平。

1.3 統(tǒng)計分析

2 結(jié)果

2.1 RHR模型構(gòu)建及血壓變化

大鼠在造模過程中無死亡，造模后飼養(yǎng)過程中死亡5只，其中2例死于術(shù)后大出血，3例死于腎衰竭，予以補充復(fù)制相同數(shù)量的模型。兩組的基礎(chǔ)血壓無顯著差異，動態(tài)觀察顯示手術(shù)后第2周RHR組大鼠血壓即開始升高，直至12周趨于穩(wěn)定，在飼養(yǎng)過程中假手術(shù)組大鼠的血壓無明顯變化。如圖1所示，RHR組術(shù)后2、4、6、8、10、12 周血壓均明顯高于假手術(shù)組(均P<0.05)。

2.2 血清MMP-9和TIMP-1水平

術(shù)后12周，RHR大鼠血清MMP-9高于假手術(shù)組[(783.4±109.79)比(573.4±109.59) ng/mL，P<0.05]。

注：與假手術(shù)組比較，#P<0.05。圖1 兩組大鼠12周內(nèi)不同時間點血壓變化Note.Compared with the sham group,#P<0.05.Fig.1 Changes of SBP in the rats at different time-points in 12weeks

注：與假手術(shù)組比較，#P<0.05。圖2 各組大鼠腦和主動脈MMP-9表達變化情況Note.Compared with the sham group,#P<0.05.Fig.2 Changes of the MMP-9 protein expression in the brain and vessels of rats in each group

2.3 腦和主動脈MMP-9和TIMP-1的表達

而RHR大鼠血清TIMP-1低于假手術(shù)組[(313.02±83.9)比(976.19±191.1) pg/mL，P<0.05]。術(shù)后12 周，Western blot 和免疫組化的結(jié)果證實RHR組腦和主動脈組織中MMP-9 的表達均高于假手術(shù)組(P<0.05)，見圖2～3；RHR組腦和主動脈組織中TIMP-1的表達則均低于假手術(shù)組(P<0.05)，見圖4～5。

2.4 血壓與MMP-9和TIMP-1的相關(guān)分析

剔除觀察過程中發(fā)生腦卒中大鼠，分別對收縮壓與血清、腦組織和血管中MMP-9和TIMP-1的關(guān)系進行相關(guān)分析，如圖6～8所示，收縮壓與血清及組織中MMP-9表達呈正相關(guān)(r=0.557，r=0.774和r=0.661，均P<0.05)，與血清及組織中TIMP-1表達呈負相關(guān)(r=-0.481，r=-0.535和r=-0.685，均P<0.01)。

注：與假手術(shù)組比較，#P<0.05。腦標(biāo)尺：20 μm，血管標(biāo)尺：200 μm。圖3 各組大鼠腦和主動脈MMP-9免疫組化(×400)Note.Compared with the sham group,#P<0.05. Scale bar=20 μm for brain and 200 μm for blood vessels.Fig.3 Changes of MMP-9 expression and distribution in the brain and vessels of rats in each group

注：與RHR組比較，#P<0.05。圖4 各組大鼠腦和主動脈TIMP-1表達情況Note.Compared with the RHR group,#P<0.05.Fig.4 Changes of TIMP-1 protein expression in the brain and vessels of rats in each group

注：與RHR組比較，★P<0.05，腦標(biāo)尺：20 μm，血管標(biāo)尺：200 μm。圖5 各組大鼠腦和主動脈TIMP-1免疫組化Note.Compared with the RHR group,★P<0.05.Scale bar=20 μm for brain and 200 μm for vessels.Fig.5 Changes of the TIMP-1 expression and distribution in the brain and vessels of rats in each group(Immunohistochemical staining)

圖6 RHR組大鼠術(shù)后12 周收縮壓與血清MMP-9和TIMP-1水平的相關(guān)分析Fig.6 Correlation analysis between the systolic blood pressure and serum MMP-9 and TIMP-1 levels in the rats of RHR group

圖7 RHR組大鼠術(shù)后12 周收縮壓血壓與腦及主動脈MMP-9蛋白表達的相關(guān)分析Fig.7 Correlation analysis between the systolic blood pressure and brain and vascular MMP-9 protein expression in the rats of RHR group

圖8 RHR組大鼠術(shù)后12 周收縮壓血壓與腦及主動脈TIMP-1蛋白表達的相關(guān)分析Fig.8 Correlation analysis between the systolic blood pressure and brain and vascular TIMP-1 protein expression in the rats of RHR group

3 討論

高血壓是心腦血管疾病最重要的危險因素。MMP-9 是基質(zhì)金屬蛋白酶系家族中降解細胞外基質(zhì)(ECM)的主要蛋白溶解酶，具有降解I、IV、V型膠原及明膠、軟骨蛋白多糖、層粘蛋白、纖維連接蛋白等能力。MMP-9參與了高血壓性血管重塑過程[9]。此外，MMP-9 在動脈粥樣硬化的形成、發(fā)展和破裂過程中發(fā)揮關(guān)鍵作用[10, 11]。MMP-9 還與動脈粥樣硬化斑塊內(nèi)出血有關(guān)[12]。TIMP-1是MMP-9 主要的內(nèi)源性抑制劑[7]，在小鼠模型中通過升高TIMP-1可減輕動脈粥樣硬化的進展[13]。研究證實，MMP-9在腦動脈瘤的發(fā)展過程中發(fā)揮關(guān)鍵作用，通過抑制MMP-9 的基因表達可抑制彈性蛋白酶誘導(dǎo)的腦動脈瘤發(fā)生[14]。滅活TIMP-1 的基因可促進動脈瘤形成[15]，而促進局部TIMP-1 表達可阻止動脈瘤進展和破裂[16]。最近研究證實MMP-9通過破壞腦血管的基底膜導(dǎo)致血管破裂參與高血壓腦出血的發(fā)病過程[17, 18]。

本研究發(fā)現(xiàn)在高血壓大鼠模型的血清、腦和主動脈中MMP-9水平明顯升高。這與既往研究發(fā)現(xiàn)高血壓大鼠腦及血清中MMP-9升高的結(jié)果相一致[19]。在AngII 誘導(dǎo)的高血壓模型中，在傳導(dǎo)血管MMP-9的活性增高[20]。具體機制可能是通過管腔內(nèi)高壓力、氧化應(yīng)激等機制實現(xiàn)的[21]。同時，研究發(fā)現(xiàn)RHR模型血清、腦和主動脈組織中TIMP-1水平均降低。既往研究發(fā)現(xiàn)高血壓患者血液TIMP-1降低[22,23]。相關(guān)性分析發(fā)現(xiàn)收縮壓與血清、腦及主動脈組織中MMP-9表達均呈正相關(guān)，說明持續(xù)的高血壓可能誘導(dǎo)MMP-9表達，升高的MMP-9可能參與高血壓大鼠腦和血管的病理損害過程。同時，相關(guān)性分析發(fā)現(xiàn)收縮壓與血清、腦及主動脈組織中TIMP-1表達均呈負相關(guān)，表明持續(xù)的高血壓可誘導(dǎo)TIMP-1表達降低。上述結(jié)果表明高血壓可誘導(dǎo)MMP-9/TIMP-1在血液和組織的表達失衡，從而導(dǎo)致MMP-9的凈效益升高，兩者的失衡可能與高血壓性心腦血管病的病理生理過程有關(guān)。兩種標(biāo)志物在血液和組織中的變化趨勢一致，關(guān)于兩個標(biāo)志物在血液與組織之間是否存在交互關(guān)系需要進一步深入研究。

在臨床上，心腦血管疾病的患者會服用很多藥物，那么是否有藥物會影響MMP-9和TIMP-1的水平呢？研究發(fā)現(xiàn)在眾多的抗高血壓藥物中，氨氯地平[24]、依那普利[25]和氯沙坦[26]可降低MMP-9。而非洛地平和地爾硫卓則對MMP-9無影響[27]；樂卡地平[28]、依那普利[23]、賴諾普利[22]和氯沙坦[23]對TIMP-1無影響，而坎地沙坦不僅可降低MMP-9而且可升高TIMP-1[22]。他汀類藥物是心腦血管疾病防治的常用藥物，研究發(fā)現(xiàn)他汀可下調(diào)MMP-9的表達[29]。這為我們的臨床用藥選擇提供了一定的參考。

本研究發(fā)現(xiàn)高血壓大鼠血液和組織中MMP-9/TIMP-1表達失衡，MMP-9/TIMP-1失衡可能與高血壓及相關(guān)心腦血管疾病有關(guān)。MMP-9和TIMP-1可能成為相關(guān)的藥物干預(yù)靶點，下調(diào)MMP-9和升高TIMP-1的藥物可能會發(fā)揮保護作用。MMP-9和TIMP-1在血液和組織中的變化趨勢保持一致，因此，血液中MMP-9和TIMP-1的變化有可能被用作評估高血壓及相關(guān)心腦血管疾病發(fā)病風(fēng)險的生物標(biāo)志物，這使兩者具備臨床應(yīng)用前景。本文的不足之處在于未能動態(tài)檢測術(shù)后血壓與血清MMP-9/TIMP-1變化的相關(guān)性，從而使論據(jù)更具說服力，這需要在今后的實驗中進一步驗證。

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Changes of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1in serum and tissues in hypertensive rats

LI Huan-min1, LI Chun-guang2*

(1. Department of Neurology, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630,China; 2.Department of Neurology, Zhujiang Hospital of Southern Medical University,Guangzhou 510280)

Objective To investigate the expressions of metalloproteinase-9 (MMP-9)in serum, brain and aorta matrix and tissue inhibitor of metalloproteinase-1(TIMP-1) in renovascular hypertensive rats (RHR)，and to evaluate the association between blood pressure and levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1).Methods Eighty healthy male SD rats were randomly divided into RHR group (n=40) and sham-operated group (n=40). Hypertension was induced by two-kidney, two-clip (2K-2C)clamps.Systolic blood pressure (SBP) was measured every 2 weeks during 12 weeks using a tail pressure meter. Stroke was confirmed by Longa’s five-point scale and pathological examination. The expressions of MMP- 9 and TIMP-1 in the brain and aorta tissues were detected by Western blot and immunohistochemistry. The levels of serum MMP-9 and TIMP-1 were measured by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Results Compared with the sham-operated group, SBP stayed significantly elevated in the RHR group at 2, 4, 6, 8, 10 and 12weeks after the operation [(157±9.0) vs. (128±7.0), (176±10.0) vs. (122±6.0), (194±8.0)vs. (117±6.5), (202±12.0)vs. (124±8.0), (218±15.0) vs. (126.±8.5),and (224±20.0)vs. (129.±9.0) mm Hg, allP< 0.05]. 12 weeks after the surgery, the level of serum MMP-9 in the RHR group was kept significantly higher than that in the sham-operated group [(783.4±109.79)vs. (573.4±109.59) ng/mL,P<0.05],and the serum TIMP-1 level was lower in the RHR group than that in the sham-operated group[(313.02±83.9) vs. (976.19±191.1) pg/mL,P<0.05]. MMP-9 expressions were significantly higher in the brain and aorta in the RHR group than that in the sham-operated group(bothP<0.05), and TIMP-1 expressions were lower than that in the sham-operated group(bothP<0.05).The Pearson correlation analysis showed that MMP-9 levels in serum,brain and aorta were positively correlated with systolic blood pressure (r=0.557,r=0.774 andr=0.661,allP<0.05), and TIMP-1 levels were negatively correlated with systolic blood pressure(r=-0.481，r=-0.535 andr=-0.685，allP<0.01). Conclusions Hypertension induces increased MMP-9 and decreased TIMP-1 in serum, brain and aorta in renovascular hypertensive rats. There are consistent alterations of circulating and tissue MMP-9 and TIMP-1 levels in renovascular hypertensive rats. There is a relationship between increased blood pressure and high MMP-9 and low TIMP-1 in serum and tissues.

Renovascular hypertensive rats; Stroke; Matrix metalloproteinase-9; Tissue inhibitor of metalloproteinase-1

LI Chun-guang,E-mail: lichunguang2005@126.com

國家自然科學(xué)基金項目(編號：81500985)；南方醫(yī)科大學(xué)科研啟動項目(編號：PY2014N080)。

李煥敏(1980-)，女，主治醫(yī)師，碩士研究生，研究方向：神經(jīng)重癥及腦血管病。E-mail: lihuanmin_2004@126.com

李春光(1980-)，男，主治醫(yī)師，博士研究生，研究方向：腦血管病。E-mail: lichunguang2005@ 126.com

Q95-33

A

1005-4847(2017) 02-0138-08

10.3969/j.issn.1005-4847.2017.02.005

2016-09-18