靶向线粒体和过氧化物酶体载体的构建及在心肌缺氧模型中的应用  

Construction of Vectors Targeting Mitochondria and Peroxisomes and Their Application in Myocardial Hypoxia Model

作　　者：蔡蕊 吴兹航 李阳[3] 宋晓伟 宋金超 胡淑婷[1] CAI Rui;WU Zihang;LI Yang;SONG Xiaowei;SONG Jinchao;HU Shuting(School of Basic Medicine,Ningxia Medical University,Yinchuan 750004;Department of Heart Medicine,Changhai Hospital,Naval Medical University,Shanghai 200433;Department of Anesthesiology,Shidong Hospital,University of Shanghai for Science and Technology,Shanghai 200093)

机构地区：[1]宁夏医科大学基础医学院,银川750004 [2]海军军医大学附属长海医院心内科,上海200433 [3]上海理工大学附属市东医院麻醉科,上海200093

出　　处：《宁夏医科大学学报》2025年第3期233-241,共9页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81760076);杨浦区医学重点学科项目(22YPZA08)。

摘　　要：目的构建用于特异性标记线粒体和过氧化物酶体的重组腺病毒载体,并研究在缺氧条件下心肌细胞中以上两种细胞器的变化。方法通过PCR技术,构建pShuttle-Mito-EGFP和pShuttle-mCherry-Peroxisome质粒。Sanger测序检测目的片段。包装及扩增重组腺病毒,氯化铯梯度离心法纯化病毒,梯度稀释法确定病毒滴度。体外分离并提取的SD乳鼠原代心肌细胞(rat cardiomyocytes,RCM),分别感染pAd-Mito-EGFP和pAd-mCherry-Peroxisome病毒24 h后,验证其是否能够特异性地标记目标细胞器。RCM分别感染pAd-Mito-EGFP和pAd-mCherry-Peroxisome病毒,分为常氧组和缺氧组,处理24 h后,观察线粒体和过氧化物酶体分布的变化。同时感染两种病毒的RCM也进行同样处理,观察细胞器数量、形态及位置关系的变化。结果DNA测序验证pAd-Mito-EGFP和pAd-mCherry-Peroxisome重组腺病毒构建成功。经测定,重组腺病毒的滴度约为9×10^(8)PFU·mL^(-1)。荧光标记可以正确标记细胞质中网格状线粒体和点状分布的过氧化物酶体,与常氧培养条件下相比,缺氧刺激后线粒体出现碎片化且丰度降低(P均<0.05),单个细胞中过氧化物酶体数量减少(P<0.01)。计算皮尔森相关系数(PCC)和曼德斯共定位系数(MCC)显示,与常氧培养条件下相比,缺氧刺激后两种细胞器相关系数和重叠值均减小(P均<0.05)。结论成功构建特异性标记线粒体和过氧化物酶体的腺病毒载体,心肌缺氧后这两种细胞器的丰度减少且位置关系减弱。Objective To construct recombinant adenoviral vectors for the specific labeling of mitochondria and peroxisomes,and to investigate the changes in these two organelles in cardiomyocytes under hypoxic conditions.Methods Using PCR technology to construct the pShuttle-Mito-EGFP and pShuttle-mCherry-Peroxisome plasmids.Primary cardiomyocytes from SD rats(rat cardiomyocytes,RCM)were isolated and infected with pAd-Mito-EGFP and pAd-mCherry-Peroxisome viruses to verify the specific labeling of target organelles.RCM were infected with either pAd-Mito-EGFP or pAd-mCherry-Peroxisome viruses and divided into normoxic group and hypoxic group.After 24 hours of treatment,changes in the distribution of mitochondria and peroxisomes were observed.Additionally,RCM infected with both viruses were treated similarly to assess changes in the number,morphology,and spatial relationships of the organelles.Results DNA sequencing confirmed the successful construction of pAd-Mito-EGFP and pAd-mCherry-Peroxisome recombinant adenoviruses.The viral titer was determined to be approximately 9×10^(8)PFU·mL^(-1).Fluorescence labeling correctly identified the network-like distribution of mitochondria and punctate peroxisomes in the cytoplasm.Compared to normoxic conditions,mitochondria were fragmented and their abundance decreased under hypoxic conditions(P all<0.05),and the number of peroxisomes per cell decreased(P<0.01).The calculation of Pearson correlation coefficient(PCC)and Manders’overlap coefficient(MCC)showed that,compared to normoxic conditions,hypoxic stimulation resulted in decreased correlation and overlap between the two organelles(P all<0.05).Conclusion The recombinant adenoviral vectors for specific labeling of mitochondria and peroxisomes were successfully constructed.The preliminary findings suggest that,after myocardial hypoxia,the abundance of both organelles decreases,and their spatial relationship weakens.

关 键 词：线粒体 过氧化物酶体 心肌细胞 缺氧 

分 类 号：R541.6[医药卫生—心血管疾病]