摘要
目的探讨过表达Bax抑制剂1(BI-1)基因对缺血再灌注损伤的影响及其相关机制。方法30只SD大鼠随机分为假手术组、缺血再灌注组(I/R组)、对照的腺病毒载体组(Ad-EGFP组)、过表达BI-1的腺病毒载体组(Ad-BI-1组)、环孢素A(CsA组)。建立大鼠心肌缺血再灌注损伤(MIRI)模型后,TTC染色观察各组大鼠心肌梗死面积,TUNEL染色观察心肌细胞凋亡情况,电镜下观察心肌细胞超微结构变化,qRT-PCR检测各组大鼠心肌组织中BI-1 mRNA表达。分离培养乳鼠心肌细胞并按细胞处理方式的不同将其分为对照组、缺氧复氧组(H/R组)、对照的腺病毒载体组(Ad-EGFP组)、过表达BI-1的腺病毒载体组(Ad-BI-1组)、环孢素A(CsA组),免疫荧光法检测BI-1的亚细胞定位,Western blot检测各组细胞中BI-1蛋白表达,Calcein-AM法检测各组心肌细胞线粒体通透性转换孔(MPTP)开放水平;应用Western blot检测凋亡相关蛋白Bcl-2、Bax、细胞色素C(CytC)及Caspase-3、Caspase-9的表达。结果缺血再灌注后,与假手术组相比,I/R组、Ad-EGFP组、Ad-BI-1组、CsA组大鼠心肌梗死面积显著增加,心肌细胞凋亡数明显增多,心肌线粒体结构受损显著加重(P<0.05);而与I/R组或Ad-EGFP组相比,Ad-BI-1组、CsA组大鼠的上述指标明显改善(P<0.05)。qRT-PCR结果显示,与假手术组相比,I/R组、Ad-EGFP组、CsA组中BI-1 mRNA表达均显著降低,而Ad-BI-1组BI-1 mRNA表达明显增加(P<0.05)。成功分离乳鼠心肌细胞,免疫细胞结果显示BI-1主要定位于心肌细胞内质网;与对照组相比,H/R组、Ad-EGFP组、Ad-BI-1组及CsA组的Calcein-AM荧光强度显著降低(P<0.05),而Ad-BI-1组及CsA组较H/R组细胞明显增加(P<0.05);Western blot结果显示,H/R组、Ad-EGFP组、CsA组中BI-1蛋白表达较对照组显著降低(P<0.05),而Ad-BI-1组BI-1蛋白表达明显增加(P<0.05)。此外,在大鼠心肌组织中,与假手术组相比,I/R组、Ad-EGFP组、Ad-BI-1组与CsA组大鼠的Bcl-2/Bax比值均显著�
Aim To investigate the effect of overexpression of Bax inhibitor-1(BI-1)gene in cardiomyocytes on ischemia-reperfusion injury and its mechanism.Methods Thirty SD rats were randomly divided into sham operation group(sham group),ischemia-reperfusion group(I/R group),adenovirus control group(Ad-EGFP group),overexpression of BI-1 genome group(Ad-BI-1 group),cyclosporine A group(CsA group).After the establishment of myocardial ischemia-reperfusion injury(MIRI)model in rats,TTC staining was used to observe myocardial infarction area,TUNEL staining was used to observe myocardial apoptosis,ultrastructural changes of myocardial cells were observed under electron microscope;qRT-PCR was used to detect the expression of BI-1 mRNA in the myocardium of rats in each group.The myocardial cells of neonatal rats were isolated and cultured and divided into control group,hypoxia/reoxygenation group(H/R group),adenovirus control group(Ad-EGFP group),overexpression of BI-1 genome group(Ad-BI-1 group)and cyclosporine A group(CsA group)according to different cell treatment.The subcellular localization of BI-1 was detected by immunocytochemistry,the expression of BI-1 protein was detected by Western blot,and the MPTP opening level of myocardial mitochondria was detected by Calcein-AM;and the expression of apoptosis related proteins Bcl-2,Bax,CytC,Caspase-3 and Caspase-9 were detected by Western blot.Results Compared with sham group,myocardial infarct area,apoptosis number,mitochondrial structure damage in I/R group,Ad-EGFP group,Ad-BI-1 group and CSA group increased significantly(P<0.05),while those in Ad-BI-1 group and CsA group decreased significantly(P<0.05)compared with I/R group.qRT-PCR results showed that compared with sham group,BI-1 mRNA expression decreased significantly in I/R group,Ad-EGFP group and CsA group,while it increased significantly in Ad-BI-1 group;Subcellular location showed that BI-1 was mainly located in the endoplasmic reticulum of cardiomyocytes,and the fluorescence intensity of Calcein-AM was significantly lowe
作者
钟小兰
班努·库肯
景江新
ZHONG Xiaolan;Bannu·kuken;JING Jiangxin(Department of Cardiology,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang 830063,China)
出处
《中国动脉硬化杂志》
CAS
2021年第3期222-231,共10页
Chinese Journal of Arteriosclerosis
基金
新疆维吾尔自治区自然科学基金项目(2017D01C248)。
