摘要
目的应用突变型的甲硫酰基-tRNA合成酶(MetRS^(L247G))标记新生蛋白技术特异性提取和分析骨髓间充质干细胞(BMSCs)移植于缺血心脏后的新生蛋白质组,以探究移植后BMSCs可能的作用机制。方法通过慢病毒感染的方法使BMSCs的MetRS基因第274位进行点突变,实现BMSCs新生蛋白的叠氮左亮氨酸(ANL)标记,并采用荧光非标准氨基酸标记(FUNCAT)验证此技术对BMSCs新生蛋白的标记效果。将健康雌性8~10周龄30只C57BL/6J小鼠分为实验组(n=15)与对照组(n=15)。实验组以结扎冠状动脉前降支的方法构建急性心肌梗死小鼠模型,对照组小鼠仅行开胸手术,不进行左前降支冠状动脉结扎。造模后两组小鼠均注射MetRS^(L247G)基因突变的BMSCs(MetRS^(L247G)-BMSCs),并在梗死区旁缺血区域的3个不同部位进行心肌注射。两组小鼠分别在术后第0、2、6天(每个时间点n=5)腹腔注射ANL,1次/6 h,共4次,24 h后实施安乐死,并分离心脏组织。采用针对ANL标记蛋白的富集技术及液相色谱-串联质谱(LC-MS)分析,收集移植于两组小鼠心肌后BMSCs所合成且被标记的新生蛋白。采用基因本体论(GO)分析、京都基因与基因组百科全书(KEGG)分析、蛋白质互作网络(PPI)分析及热图分析3个时间点的差异蛋白,并筛选出关键通路及基因。结果荧光显微镜下可见MetRS^(L247G)慢病毒感染的BMSCs标记有mCherry信号,证实MetRS^(L247G)-BMSCs细胞系构建成功,且只有同时加入MetRS^(L247G)-BMSCs和ANL的培养基中的新生蛋白标记有绿色荧光信号,验证了此标记方法的敏感性及特异性。GO分析显示,差异蛋白主要集中在细胞外泌体、细胞外基质、黏着斑等基本的细胞生物过程;KEGG分析及PPI分析显示,差异蛋白主要参与补体和凝血级联通路、肌动蛋白细胞骨架调节通路、细胞凋亡通路等过程;热图分析显示,与对照组比较,实验组小鼠移植后第1天,其心肌组织抗凋亡相关因子及细胞黏附相关因子表达均明显上调(P<0.05);实验组移植后第3天,抗凋亡相关因子、促凋亡相关因子及细胞黏附相关因子表达均明显上调(P<0.05);实验组移植后第7天,抗凋亡相关因子、细胞分化相关因子及细胞黏附相关因子表达均明显上调(P<0.05);而实验组第1、7天凋亡诱导因子1的表达明显下调(P<0.05)。此外,在第3天时大部分差异蛋白的表达均明显上调(P<0.05),如抗凋亡相关因子S100钙结合蛋白A11、簇集素、凝溶胶蛋白,促凋亡相关因子组织蛋白酶B,细胞分化相关因子转胶蛋白-2,细胞黏附相关因子丝切蛋白-1、骨膜蛋白、纤连蛋白等。结论MetRS^(L247G)突变可使BMSCs吸收ANL,使新合成的蛋白被标记,证实了该新生蛋白标记技术的可行性。BMSCs在缺血心肌组织中新生蛋白的作用主要在外泌体分泌及细胞外基质组织方面。BMSCs可能通过影响补体和凝血级联反应、激活炎症因子、调整肌动蛋白细胞骨架结构及调控细胞凋亡等途径,以适应及应对缺血缺氧的环境,从而维持BMSCs的存活。
Objective To specifically extract and analyze nascent proteins synthesized by bone marrow mesenchymal stem cells(BMSCs)after transplantation into ischemic hearts using a technique employing mutant methionyl-tRNA synthetase(MetRS^(L247G))for nascent protein labeling,in order to explore the potential mechanisms of action in BMSCs post-transplantation.Methods Point mutation at position 274 of the MetRS gene in BMSCs was induced via lentiviral infection to enable azidonorleucine(ANL)-mediated labeling of nascent proteins in BMSCs.The labeling efficiency was verified by means of fluorescent non-canonical amino-acid tagging(FUNCAT).Thirty healthy female C57BL/6J mice(8-10 weeks old)were divided into control and experimental groups,with 15 mice in each group.The acute myocardial infarction model was constructed by ligating the left anterior descending coronary artery in experimental group,while control mice underwent only thoracotomy without coronary ligation.After modeling,both groups received intramyocardial injections of MetRS^(L247G)-modified BMSCs(MetRS^(L247G)-BMSCs)at 3 different sites in the peri-infarct ischemic region.Mice were intraperitoneally injected with ANL every 6 hours for 4 times on postoperative days 0,2,and 6(n=5 for each time point)respectively,euthanized 24 h after the last injection,and cardiac tissues were isolated.The newly synthesized and labeled proteins produced by BMSCs after transplantation into the myocardium of experimental and control groups were collected,using an enrichment technique for ANL-tagged proteins and liquid chromatography-tandem mass spectrometry(LC-MS)analysis.Gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,protein-protein interaction(PPI)analysis,and heatmap visualization analysis were performed to identify differentially expressed proteins at the 3 time points and screen key pathways and genes.Results Under fluorescence microscopy,the MetRS^(L247G) lentivirus-infected BMSCs were observed to be labelled with mCherry signals,confirming the successful construction of the MetRS^(L247G)-BMSCs cell line.Green fluorescent signals were detected only in nascent proteins in culture medium containing both MetRS^(L247G)-BMSCs and ANL,validating the sensitivity and specificity of the labeling method.GO analysis revealed that differentially expressed proteins were primarily involved in basic cellular biological processes such as extracellular exosome formation,extracellular matrix organization,and focal adhesion.KEGG and PPI analyses indicated that the differential proteins were mainly involved in complement and coagulation cascade pathway,actin cytoskeleton regulation pathway,and apoptosis pathway.Heatmap analysis showed significantly upregulated expression of anti-apoptosis and cell adhesion-related factors in experimental group on day 1(P<0.05),upregulated anti-apoptotic factors,pro-apoptotic factors,and cell adhesion-related factors on day 3(P<0.05),and upregulated anti-apoptotic factors,cell differentiation-related factors,and cell adhesion-related factors on day 7(P<0.05)compared with control group.Expression of apoptosis-inducing factor 1 was significantly downregulated on days 1 and 7(P<0.05).On day 3,most differentially expressed proteins,including anti-apoptosis factors(Protein S100-A11,Clusterin,Gelsolin),pro-apoptosis factor(Cathepsin B),cell differentiation-related factor(Transgelin-2),and cell adhesion-related factors(Cofilin-1,Periostin,Fibronectin)were significantly upregulated(P<0.05).Conclusions The MetRS^(L247G) mutation enables BMSCs to incorporate ANL and synthesize labeled proteins,confirming the feasibility of this nascent protein labeling technique.Nascent proteins of BMSCs in ischemic myocardium primarily contribute to extracellular exosome secretion and extracellular matrix organization.BMSCs may adapt to and respond to ischemic and hypoxic environments by influencing complement and coagulation cascades,activating inflammatory factors,regulating actin cytoskeleton structure,and modulating apoptosis,thereby maintaining the survival of BMSCs.
作者
卢婉儿
代蓥
汤穆浛
魏康
陈术佳
黄怀
林静
彭浩荣
周礼轩
韩敦正
Lu Wan-Er;Dai Ying;Tang Mu-Han;Wei Kang;Chen Shu-Jia;Huang Huai;Lin Jing;Peng Hao-Rong;Zhou Li-Xuan;Han Dun-Zheng(Department of Cardiology,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong 510120,China;Nanshan College,Guangzhou Medical University,Guangzhou,Guangdong 511436,China;the First Clinical College,Guangzhou Medical University,Guangzhou,Guangdong 511436,China;Department of Emergency,Guangzhou Red Cross Hospital,Guangzhou,Guangdong 510220,China)
出处
《解放军医学杂志》
北大核心
2025年第8期991-998,共8页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(82100263)
广州市基础研究计划市校(院)企联合资助专题“登峰医院”项目(2025A03J4401,2024A03J0655)。
关键词
间充质干细胞
间充质干细胞移植
心肌梗死
心力衰竭
蛋白质组学
mesenchymal stem cells
mesenchymal stem cell transplantation
myocardial infarction
heart failure
proteomics
