沈俊 袁平 唐俊明 張蕾 趙繼先 張煥鑫 薛仕珍 馮怡 王家寧
基礎(chǔ)研究
中胚層同源盒基因1在急性心肌梗死大鼠心臟微血管內(nèi)皮的表達(dá)變化
沈俊 袁平 唐俊明 張蕾 趙繼先 張煥鑫 薛仕珍 馮怡 王家寧
目的 研究中胚層同源盒基因1(Meox1)在急性心肌梗死大鼠心臟微血管內(nèi)皮的表達(dá)情況。方法 成年雄性SD大鼠20只隨機(jī)分成急性心肌梗死組和假手術(shù)組,各10只。采用結(jié)扎左冠狀動脈前降支結(jié)扎法構(gòu)建急性心肌梗死模型,假手術(shù)組僅在左冠狀動脈前降支穿線不結(jié)扎。建模后將急性心肌梗死組作為實驗組,假手術(shù)組作為對照組。建模后第7天處死兩組大鼠,進(jìn)行免疫組化染色,分析急性心肌梗死后心臟微血管上Meox1的表達(dá)情況。結(jié)果 通過免疫組化分析發(fā)現(xiàn),在心肌梗死區(qū)及梗死邊緣區(qū)微血管內(nèi)皮可見棕黃色顆粒沉積,提示有Meox1表達(dá);在假手術(shù)組及梗死遠(yuǎn)離區(qū)微血管內(nèi)皮上未見Meox1的表達(dá)。結(jié)論 在急性心肌梗死大鼠心臟微血管內(nèi)皮上Meox1表達(dá)增多,推測其可能參與心肌梗死后的血管重塑。
心肌梗死; 同源盒基因Meox1; 表達(dá)
心肌梗死后心肌纖維化的發(fā)生,導(dǎo)致心臟房室結(jié)構(gòu)改變,引起心肌病理性重構(gòu),最終引起心功能衰竭甚至患者死亡[1-4],因此,遏制心肌梗死后心肌纖維化的發(fā)生就顯得尤為重要。盡管目前有許多方法試圖從不同角度來解決這一難題,然而臨床效果均不理想[5-7]。近年來基于基因和干細(xì)胞手段防治心血管疾病效果顯著,因此,尋求有效基因治療靶點(diǎn)非常必要[8-10]。前期研究表明,中胚層同源盒基因1(Meox1)是控制發(fā)育的主要基因,參與心血管系統(tǒng)的發(fā)育及心血管疾病的發(fā)生[11,12]。本研究通過制備心肌梗死模型,利用免疫組化觀察心肌梗死后Meox1的表達(dá)情況。
1.1 實驗動物、主要材料與儀器 SPF級雄性SD大鼠20只,體重200~230 g,由湖北醫(yī)藥學(xué)院實驗動物中心[SCXK(鄂)2011-0008]提供。飼養(yǎng)于屏障設(shè)施[SYXK(鄂)2011-0031]。顯微手術(shù)器械購于美國Fine science公司,6-0無菌醫(yī)用非吸收性縫合線、無損傷縫合針購于杭州富陽醫(yī)用縫合針線廠。RM2245型病理切片機(jī)、H11220型烘片機(jī)、H11210型攤片機(jī)購于德國Leica公司,BX-51型光學(xué)顯微鏡購于日本Nikon公司。Meox1多克隆抗體購于美國abcom公司,DAB顯色試劑盒購于武漢博士德公司。1.2 方法
1.2.1 心肌梗死動物模型的構(gòu)建 將20只大鼠隨機(jī)分為2組:心肌梗死組10只,假手術(shù)組10只。心肌梗死模型按照Kumar等[13]的改進(jìn)方法,采用配制的10%水合氯醛(300 mg/kg)對大鼠進(jìn)行腹腔注射麻醉,氣管插管接小動物專用呼吸機(jī)輔助通氣。在胸骨左緣3~4肋間開胸,鈍性分離皮下組織及肌層,顯露心臟,在心臟收縮瞬間迅速將心臟擠出,用6-0號非吸收無損傷縫合線結(jié)扎冠狀動脈前降支,當(dāng)結(jié)扎部位以下心肌顏色變白、心電圖顯示相應(yīng)ST段弓背抬高,表示造模成功?;丶{心臟并縫合胸壁。假手術(shù)組手術(shù)過程同心肌梗死組,但僅在左冠狀動脈前降支穿線,不做結(jié)扎。術(shù)后3 d大鼠肌肉注射青霉素以防感染。建模成功后7 d,麻醉并處死大鼠,取心室肌置于4%多聚甲醛固定過夜,常規(guī)脫水、透明、浸蠟、包埋等處理。切厚度為5μm組織切片,并編號。
1.2.2 免疫組化分析 切片常規(guī)脫蠟、水化后微波修復(fù)抗原,3%H2O2孵育,10%馬血清封閉。一抗使用Meox1兔多克隆抗體(1∶100),二抗使用鼠抗兔多克隆抗體(1∶100),按照ABC試劑盒進(jìn)行DAB染色(現(xiàn)配現(xiàn)用),蘇木精染色,鹽酸-酒精分化,肥皂水返藍(lán),乙醇脫水,二甲苯透明,中性樹脂封片。
Meox1在心臟微血管內(nèi)皮表達(dá)情況 假手術(shù)組(圖1D)及梗死遠(yuǎn)離區(qū)(圖1C)微血管內(nèi)皮未見Meox1的表達(dá);梗死邊緣區(qū)(圖1A)及梗死區(qū)(圖1B)微血管內(nèi)皮可見Mexo1表達(dá),其動態(tài)變化可能在心肌梗死后血管重構(gòu)中起作用。
急性心肌梗死后多半有慢性心功能不全,嚴(yán)重影響生存質(zhì)量。如何有效防止心肌梗死后心肌纖維化,預(yù)防心室重構(gòu),迫在眉睫。目前研究表明,心肌梗死后心肌纖維化發(fā)生與RAAS系統(tǒng)異常激活、細(xì)胞外基質(zhì)異常沉積、各種轉(zhuǎn)錄生長因子及基因激活相關(guān)。
同源盒基因(homeobox gene)最初在果蠅中發(fā)現(xiàn),是控制發(fā)育的主要基因,對動物的胚胎發(fā)育和細(xì)胞生長、分化的調(diào)控起關(guān)鍵作用[14]。同源盒蛋白在中胚層和間充質(zhì)的大范圍內(nèi)廣泛表達(dá)[15]。所編碼的蛋白質(zhì)可能在調(diào)節(jié)體節(jié)發(fā)育的分子信號網(wǎng)絡(luò)中發(fā)揮作用[16,17];在內(nèi)皮細(xì)胞Meox激活CDKN1A和CDKN2A的表達(dá),調(diào)節(jié)血管細(xì)胞增殖。雖然它以DNA依賴的方式激活CDKN1A,但卻以DNA非依賴的方式激活CDKN2A[18]。Meox家族共有兩個成員,包括 Meox1(mesoderm/mesenchyme homeobox gene1)和 Meox2(mesoderm/mesenchyme homeobox gene 2)[19]。近年來,Meox2在心血管領(lǐng)域研究比較多,然而有關(guān)Meox1在心血管系統(tǒng)的作用知之甚少。王書美等[20]通過轉(zhuǎn)基因小鼠,發(fā)現(xiàn)心臟Meox1過表達(dá)引起擴(kuò)張型心肌病。Gianakopoulos等通過P19胚胎癌細(xì)胞建立Meox1過表達(dá)的細(xì)胞系,研究發(fā)現(xiàn)Meox1可誘導(dǎo)心臟的形態(tài)發(fā)生變化[21]。
本研究結(jié)果表明,大鼠急性心肌梗死后,在梗死區(qū)及梗死邊緣區(qū)微血管內(nèi)皮中可見Meox1表達(dá),而遠(yuǎn)離區(qū)及正常微血管內(nèi)皮未見其表達(dá)。我們據(jù)此推測:在大鼠急性心肌梗死后,梗死區(qū)及梗死邊緣區(qū)缺血缺氧,誘導(dǎo)Meox1表達(dá)增多,提示其參與心肌梗死后的血管重構(gòu),然而具體機(jī)制有待深入研究,以期為臨床提供新的治療靶點(diǎn)。
(本文圖片見后插二)
[1]Mavrogeni S,Markousis-Mavrogenis G,Koutsogeorgopoulou L,et al.Cardiovascular magnetic resonance imaging pattern at the time of diagnosis of treatment naive patients with connective tissue diseases.Int J Cardiol,2017,236:151-156.
[2]Hervas A,Ruiz-Sauri A,Gavara J,et al.A Multidisciplinary Assessment of Remote Myocardial Fibrosis After Reperfused Myocardial Infarction in Swine and Patients.J Cardiovasc Transl Res,2016,9:321-333.
[3]Shen XC,YP,Xiao TT,et al.Protective effect of oxymatrine on myocardial fibrosis induced by acute myocardial infarction in rats involved in TGF-β-Smads signal pathway.J Asian Nat ProdRes,2011,13:215-224.
[4]Wang X,Lv H,Gu Y,et al.Protective effect of lycopene on cardiac function and myocardial fibrosis after acute myocardial infarction in rats via the modulation of p38 and MMP-9.J Mol Histol,2014,45:113-120.
[5]Hartford M,Karlson BW,Sjolin M,et al.Symptoms,thoughts,and environmental factors in suspected acute myocardial infarction.Heart Lung,2015,22:64-70.
[6]Yang CC,Yip HK,Chen KH,et al.Impact of impaired cardiac function on the progression of chronic kidney disease——role of pharmacomodulation of valsartan.Am J Transl Res,2017,9:2548-2566.
[7]Cvjeticanin B,Prutki M,Dumiccule I,et al.Possible target for preventing fibrotic scar formation following acute myocardial infarction.Med Hypotheses,2014,83:656-658.
[8]沈祥春,楊鈺萍,徐旖旎,等.基于TGF-β-Smads信號的氧化苦參堿干預(yù)急性心肌梗死誘發(fā)實驗性大鼠心肌纖維化的研究.中國中藥雜志,2012,37:632-636.
[9]Fan DL,Zhao WJ,Wang YX,et al.Oxymatrine inhibits collagen synthesis in keloid fibroblasts via inhibition of transforming growth factor-β1/Smad signaling pathway.IntJDermatol,2012,51:463-472.
[10]Teekakirikul P,Eminaga S,Toka O,et al.Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by nonmyocyteproliferation and requiresTgf-β.JClin Invest,2010,120:3520-3529.
[11]Liu Q,Jingwei T,Xu Y,et al.Protective effect of RA on myocardial infarction induced-cardiac fibrosis via AT1R/p38 MAPK pathway signaling and modulation of the ACE2/ACE ratio.J Agric Food Chem,2016,64:6716-6722.
[12]Nguyen PD,Hollway GE,Sonntag C,et al.Haematopoietic stem cellinduction by somite-derived endothelialcells controlled by meox1.Nature,2014,512:314-318.
[13]Kumar M,Kasala ER,Bodduluru LN,et al.Animal models of myocardial infarction:Mainstay in clinical translation.Regul Toxicol Pharmacol,2016,76:221-230.
[14]Mohamed JY,F(xiàn)aqeih E,Alsiddiky A,et al.Mutations in MEOX1,Encoding Mesenchyme Homeobox 1,Cause Klippel-Feil Anomaly.Am J Hum Genet,2013,92:157-61.
[15]Dong K,Hsu A,Chen S.Meox1,a Novel Regulator in Tgf-βinduced Smooth Muscle Cell Differentiation.Arterioscl Throm Vas,2013,33:A544.
[16]Bayrakli F,Guclu B,Yakicier C,et al.Mutation in MEOX1 gene causes a recessive Klippel-Feil syndrome subtype.BMC Genetics,2013,14:1-7.
[17]Rosti R.Of mice,men,and King Tut:autosomal recessive Klippel-Feil syndrome is caused by mutations in MEOX1. Clinical Genetics,2013,84:19.
[18]Douville JM,Cheung DY,Herbert KL,et al.Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.PLoS One,2011,6:e29099.
[19]Gasparyan M,Burnett JP,Sun L,et al.Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem cells.Cancer Res,2016,76:2509.
[20]王書美,呂丹,陳煒,等.Meox1在心臟過表達(dá)引起轉(zhuǎn)基因小鼠擴(kuò)張性心肌病.中國比較醫(yī)學(xué)雜志,2010,20:9-13+18.
[21]Gianakopoulos PJ,Skerjanc IS.Hedgehog signaling induces cardiomyogenesis in P19 cells.Biol Chem,2005,280:21022-21028.
Expression of mesodermal homobox gene 1 in cardiac microvascular endothelial cells in rats with acute myocardial infarction
SHEN Jun*,YUAN Ping,TANG Jun-ming,et al.*Department of Cardiology,Renmin Hospital,Hubei University of Medicine,Shiyan 442000,China Corresponding author:WANG Jia-ning,E-mail:rywjn@vip.163.com
Objective To study the expression of mesoderm homebox gene 1(Meox1)in the microvascular endothelium of acute myocardial infarction rats.MethodsTwenty adult male SD rats were randomly divided into acute myocardial infarction group and sham operation group.The model of acute myocardial infarction was established by ligating the left anterior descending coronary artery.The sham operation group was not ligated only in the anterior descending branch of the left coronary artery.The acute myocardial infarction group was used as the experimental group and the sham operation group as the control group.Two groups of rats were sacrificed on the 7th day after modeling.Immunohistochemical staining was performed to analyze the expression of Meox1 on the cardiac microvessels after acute myocardial infarction.ResultsThe expression of Meox1 was observed by immunohistochemical analysis in the microvascular endothelium in the infarcted and infarcted areas,suggesting that Meox1 expression was not found in the sham operation group and the infarcted lesion.ConclusionThe expression of Meox1 in the heart of patients with acute myocardial infarction increased,suggesting that it may be involved in vascular remodeling after myocardial infarction.
Myocardial infarction; Mesoderm homebox gene 1; Expression
10.3969/j.issn.1672-5301.2017.08.023
Q95-33;R542.2+2
A
1672-5301(2017)08-0757-03
2017-02-16)
國家自然科學(xué)基金(項目編號:81270221)
442000 湖北省十堰市,湖北醫(yī)藥學(xué)院附屬人民醫(yī)院心內(nèi)科1病區(qū)(沈俊、袁平、趙繼先、張煥鑫、薛仕珍、馮怡、王家寧),臨床醫(yī)學(xué)研究所(沈俊、唐俊明、張蕾、王家寧)
王家寧,E-mail:rywjn@vip.163.com