低氧预处理对低温缺氧-复氧心肌细胞MMP-2和Cx43表达的影响  

Effects of hypoxic preconditioned on expression of MMP-2 and Cx43 in hypothermic hypoxiareoxygenation cardiomyocytes

作　　者：严旭 高鸿 柏雪 安丽 吴学艳 胡廷菊 陈锐 宋雨婷 YAN Xu;GAO Hong;BAI Xue;AN Li;WU Xueyan;HU Tingju;CHEN Rui;SONG Yuting(School of Anesthesiology,Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Department of Anaesthesia,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学麻醉学院,贵州贵阳550004 [2]贵州医科大学附属医院麻醉科,贵州贵阳550004

出　　处：《解放军医学院学报》2024年第9期969-976,983,共9页Academic Journal of Chinese PLA Medical School

基　　金：贵州省卫生健康委科学技术基金项目(Gzwkj2021-270,Gzwkj2022-120)。

摘　　要：背景 再灌注心律失常(reperfusion arrhythmia,RA)是体外循环心内直视手术中心肌缺血再灌注损伤较为常见的并发症,探索其发生机制对预防RA有重要意义。目的 研究低氧预处理H9C2细胞条件培养基对低温缺氧-复氧(hypoxia/reoxygenation,H/R)心肌细胞的保护作用,为预防和减少RA的发生提供实验支持。方法 将H9C2细胞随机分为5组,分别为阴性对照组(C组,以等体积含10%胎牛血清的DMEM培养基培养正常心肌细胞)、低氧组(H组,低氧条件培养基培养低温H/R心肌细胞)、常氧组(N组,常氧条件培养基培养低温H/R心肌细胞)、重组蛋白基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)组(MMP-2组,200 ng/mL重组蛋白MMP-2培养基培养低温H/R心肌细胞)和MMP-2抑制剂组(ARP-100组,0.03 mol/L ARP-100培养基培养低温H/R心肌细胞)。CCK-8法检测心肌细胞活力;Hoechst33342染色及流式细胞术Annexin V/PI双染检测心肌细胞凋亡率;划痕标记染料示踪技术检测缝隙连接通讯;明胶酶谱法检测细胞培养液中MMP-2活性;Western blot检测MMP-2、缝隙连接蛋白43 (connexin 43,Cx43)和磷酸化Cx43 (p-Cx43)蛋白表达;免疫荧光检测心肌细胞膜上Cx43表达。结果 与C组相比,N组心肌细胞活力降低(P<0.01),凋亡增加(P<0.01),缝隙连接通讯减弱,MMP-2表达上调(P<0.01),Cx43和p-Cx43表达均下调(P<0.01),心肌细胞膜上Cx43荧光强度减弱(P<0.05)。与N组相比,H组心肌细胞活力升高(P<0.01),凋亡减少(P<0.01),缝隙连接通讯增强,MMP-2活性降低(P<0.05),MMP-2表达下调(P<0.01),Cx43和p-Cx43表达均上调(P<0.01),心肌细胞膜上Cx43荧光强度增强(P<0.05)。与MMP-2组相比,H组和ARP-100组心肌细胞Cx43及p-Cx43表达均上调(P<0.01),心肌细胞膜上Cx43荧光强度增强(P<0.01),缝隙连接通讯增强。结论 低氧预处理H9C2细胞条件培养基通过抑制MMP-2活性介导上调Cx43和p-Cx43表达,从而改善低温H/R心肌细胞间缝隙连接通讯,减少心肌细胞�Background Reperfusion arrhythmia(RA)is a relatively common complication of myocardial ischaemia-reperfusion injury during extracorporeal circulation endocardial direct vision surgery,and it is of great significance to explore the pathogenesis of RA.Objective To investigate the protective effect of hypoxic preconditioned H9C2 cell conditioned medium on hypothermic hypoxia-reoxygenation H9C2 cardiomyocytes,so as to provide experimental support for the reduction of RA.Methods H9C2 cells were randomly divided into five groups,negative control group(equal volume of DMEM medium containing 10%fetal bovine serum cultured normal cardiomyocytes,Group C),hypoxia group(hypoxic conditioned medium cultured with hypothermic H/R cardiomyocytes,Group H),normoxia group(normoxic conditioned medium cultured with hypothermic H/R cardiomyocytes,Group N),recombinant protein MMP-2 group(200 ng/mL recombinant protein MMP-2 medium cultured with hypothermic H/R cardiomyocytes,MMP-2 group),and MMP-2 inhibitor group(0.03 mol/L ARP-100 medium cultured with hypothermic H/R cardiomyocytes,ARP-100 group).Cardiomyocyte viability was detected by CCK8 assay;cardiomyocyte apoptosis rate was detected by Hoechst33342 staining and Annexin V/PI double staining by flow cytometry;gap junction intercellular communication was detected by scrape-loading dye transfer technique;MMP-2 activity was detected by gelatin zymography in the cell culture medium;MMP-2,Cx43 and p-Cx43 protein expression were detected by Western blot;and the expression of Cx43 on the cardiomyocyte membranes was detected by immunofluorescence.Results Compared with the group C,cardiomyocyte viability in the N group was decreased(P<0.01),apoptosis was increased(P<0.01),gap junction intercellular communication was attenuated,MMP-2 expression was upregulated(P<0.01),while both Cx43 and p-Cx43 expression were downregulated(P<0.01),and Cx43 fluorescence intensity was attenuated on cardiomyocyte membranes(P<0.05).Compared with the N group,cardiomyocyte viability in the H group was elevated(P<0.

关 键 词：低氧预处理 条件培养基 细胞间缝隙连接通讯 缝隙连接蛋白43 基质金属蛋白酶-2 

分 类 号：R54[医药卫生—心血管疾病] R318[医药卫生—内科学]