TFPI对大鼠心肌缺血再灌注损伤的影响及其机制初探  

Effect of TFPI on myocardial ischemia reperfusion injury in rats and its mechanism

作　　者：李嘉舒 高薇[2] 代悦 徐睿 沈丽[1] 闫汝楠 刘越[1] 陈文佳[1] 刘文秀[1] 傅羽[1] LI Jiashu;GAO Wei;DAI Yue;XU Rui;SHEN Li;YAN Runan;LIU Yue;CHEN Wenjia;LIU Wenxiu;FU Yu(Department of Cardiology,the First Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150001,China;Department of Thyroid Surgery,the First Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150001,China)

机构地区：[1]哈尔滨医科大学附属第一医院心内科,黑龙江省哈尔滨市150001 [2]哈尔滨医科大学附属第一医院甲状腺外科,黑龙江省哈尔滨市150001

出　　处：《中国动脉硬化杂志》2023年第2期93-100,共8页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81200143、81200235和82170513);黑龙江省自然科学基金项目(QC2012C015)。

摘　　要：[目的]探究组织因子途径抑制物(TFPI)对大鼠心肌缺血再灌注(I/R)及心肌细胞缺氧复氧(H/R)损伤的影响,并从心肌细胞凋亡的变化探索其机制。[方法]在体内实验中,通过SD大鼠心脏原位结扎法可逆阻断前降支建立大鼠心肌I/R模型。将大鼠随机分为对照组、I/R组和I/R+rTFPI(重组TFPI)组,再灌注后3天采用HE染色观察大鼠心肌组织形态学变化,TTC染色法评估心肌梗死区范围,扫描透射电镜观察心肌超微结构损伤情况,Western blot法检测各组大鼠心肌组织中Bcl-2、Bax和cleaved Caspase-3蛋白的表达。在体外实验中,采用胰酶消化法及差速贴壁法培养SD乳鼠原代心肌细胞,用MIC101系统模拟心肌细胞I/R损伤,缺氧2 h、复氧12 h后建立体外心肌细胞H/R模型。将心肌细胞分为对照组、H/R组和H/R+rTFPI(10μg/L)组,用CCK-8法检测心肌细胞活力,TUNEL法检测心肌细胞凋亡率,Western blot法检测心肌细胞中Bax、Bcl-2及cleaved Caspase-3蛋白的表达水平。[结果]体内实验中,成功建立大鼠在体心肌I/R模型。HE染色结果显示I/R组较对照组心肌细胞坏死程度加重,I/R+rTFPI组较I/R组心肌细胞坏死程度减低;TTC染色示I/R+rTFPI组较I/R组心肌梗死范围减少了39.76%(P<0.05);扫描透射电镜观察显示I/R组凋亡及损伤程度较对照组加重,I/R+rTFPI组凋亡及损伤较I/R组减轻;Western blot结果示,再灌注3天后I/R组心肌组织Bcl-2的表达较对照组降低了53.43%(P<0.05),Bax和cleaved Caspase-3的表达较对照组分别增加了29.05%和73.25%(P<0.05),而I/R+rTFPI组Bcl-2的表达水平较I/R组升高了55.01%(P<0.05),Bax和cleaved Caspase-3的表达水平较I/R组分别降低了13.77%和24.25%(P<0.05)。在体外实验中,CCK-8检测结果显示H/R组细胞活力较对照组下降了29.70%(P<0.05),H/R+rTFPI组细胞活力较H/R组升高了19.77%(P<0.05)。TUNEL结果显示H/R组较对照组凋亡率增加了56.76%,H/R+rTFPI组细胞凋亡率较H/R组降低了24.55%(P<0.05)。Aim To investigate the effects of tissue factor pathway inhibitor(TFPI)on cardiomyocyte apoptosis following ischemia/reperfusion(I/R)injury.Methods I/R model was established by ligating the anterior descending coronary artery in vivo.Rats were randomly divided into three groups:the control,I/R,and I/R+rTFPI groups.HE staining was used to evaluate the morphological changes of rats myocardial tissue after reperfusion for 3 days.TTC staining was used to detect the myocardial infarct size.Ultrastructural damage in cardiomyocytes was measured by transmission electron microscopy.The expression levels of Bcl-2,Bax and cleaved Caspase-3 in rats myocardial tissue were detected by Western blot analysis.Cardiomyocytes of Sprague Dawley(SD)rats were cultured by enzyme digestion and differential adherent method in vitro experiments.Cardiomyocytes I/R injury in vitro model was established by 2 h of hypoxia and 12 h of reoxygenation using MIC101 system.Cardiomyocytes were divided into control group,H/R group,and H/R+rTFPI group(10μg/L).Cell viability was detected by CCK-8 assay.Detection of cardiomyocytes apoptosis was performed by TUNEL technique.Western blot analysis was used to detect the expression of Bax,Bcl-2 and cleaved Caspase-3.Results A myocardial I/R model was successfully established in vivo.HE staining showed myocardiumin exhibited a higher degree of necrosis than that in control group,which was milder in I/R+rTFPI group than that in I/R group(P<0.05).TTC staining showed that,compared with I/R group,myocardial infarct size reduced 39.76%in I/R+rTFPI group(P<0.05).The data of transmission electron microscopy showed that the degree of apoptosis and injury was severer in I/R group than that in control group,whereas it was milder in I/R+rTFPI group than that in I/R group.Western blot detection results showed that compared with control group,the expression of Bcl-2 decreased 53.43%in I/R group(P<0.05),but the expression of Bax and cleaved Caspase-3 increased 29.05%and 73.25%respectively(P<0.05).After adding rTFPI,the e

关 键 词：组织因子途径抑制物 缺血再灌注损伤 心肌细胞 细胞凋亡 

分 类 号：R5[医药卫生—内科学]