人脐带间充质干细胞改善小鼠下肢缺血机制的代谢组学分析  

Metabolomic analysis of the mechanisms for human umbilical cord mesenchymal stem cells improving lower limb ischemia in mice

作　　者：周梦蝶 王赞 张敏 李志红 谢晓云 朱建华 ZHOU Mengdie;WANG Zan;ZHANG Min;LI Zhihong;XIE Xiaoyun;ZHU Jianhua(Department of Geriatrics,Shanghai Yangzhi Rehabilitation Hospital Affiliated to Tongji University,Shanghai 201619,China;Department of Interventional and Vascular Surgery,Shanghai Tenth People's Hospital Affiliated to Tongji University,Shanghai 200072,China;Department of General Surgery,Zhoupu Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201318,China)

机构地区：[1]同济大学附属养志康复医院老年科,上海201619 [2]同济大学附属上海市第十人民医院介入血管外科,上海200072 [3]上海健康医学院附属周浦医院普通外科,上海201318

出　　处：《中国普通外科杂志》2023年第12期1908-1918,共11页China Journal of General Surgery

基　　金：国家自然科学基金资助项目(82072024);上海市自然科学基金资助项目(18ZR1433700)。

摘　　要：背景与目的:外周血管疾病(PAD)是因各种原因所致血流灌注不足而引发的疾病,部分患者无法手术。近年来,干细胞移植开始应用于PAD的治疗,并取得了一定的效果。然而目前尚未见其作用机制涉及代谢方面的研究。因此,本研究利用液相色谱-质谱(LC-MS)代谢组学初步探讨人脐带间充质干细胞(HUCMSC)促进下肢缺血组织修复中所涉及的代谢通路与代谢分子,并重点分析酸性鞘磷脂酶(ASM)-神经酰胺(Cer)代谢途径的变化。方法:从人脐带组织中分离获得HUCMSC,培养扩增后用流式细胞术鉴定其表面分子标记。8周龄雄性小鼠(C57BL/6J)采用结扎并离断其左侧下肢股动脉、股静脉的方法制备下肢缺血模型,将造模后的小鼠随机均分为两组,分别于缺血侧下肢局部注射HUCMSC混悬液(HUCMSC组)与PBS(对照组)。分别在术后3、7、14 d,取两组小鼠缺血侧腓肠肌组织,行HE染色与Masson染色,观察两组小鼠缺血肌肉的形态学变化;行LC-MS检测,并结合KEGG数据库分析,比较两组肌肉组织的代谢组学差异。结果:分离获得的HUCMSC高表达CD105、CD90、CD73,低表达HLA-DR、CD14、CD19、CD34。形态学观察显示,与对照组相比,HUCMSC组术后7、14 d肌肉萎缩与纤维化程度均较对照组明显改善。两组术后3、7、14 d的腓肠肌样本的非靶向代谢组学检测共鉴定出687种代谢物,其中脂类相关物质占比最大(34.088%)。负离子模式下检测的差异代谢物共37种,25种上调,12种为下调;在正离子模式下检测的差异代谢物共17种,其中11种上调,6种为下调。与对照组比较,Cer明显下调(FC=0.43),且ASM/Cer通路产物磷脂酰胆碱也明显下降(FC=0.68)。合并两组术后3、7 d的腓肠肌样本的正负离子模式数据后的KEGG通路分析结果显示,差异代谢物主要涉及通路有γ-氨基丁酸能突触、精氨酸/鸟氨酸代谢、矿物质吸收、氧化磷酸化、蛋白质代谢、甘油磷脂代谢、肌动�Background and Aims:Peripheral arterial disease(PAD)is a condition characterized by insufficient blood flow due to various reasons,and some patients are not suitable for surgery.In recent years,stem cell transplantation has been used in the treatment of PAD,showing promising results.However,the metabolic mechanisms involved in its therapeutic effects remain unclear.This study was conducted to investigate the metabolic pathways and molecules involved in the repair of lower limb ischemic tissue by human umbilical cord mesenchymal stem cells(HUCMSCs)using liquid chromatography-mass spectrometry(LC-MS)metabolomics,with a specific focus on changes in the acid sphingomyelinase(ASM)-ceramide(Cer)metabolic pathway.Methods:HUCMSCs were isolated from human umbilical cord tissue,expanded in culture,and characterized using flow cytometry for surface molecular markers.A mouse model of lower limb ischemia was created by ligating and excising the left femoral artery and vein in 8-week-old male C57BL/6J mice.Then,the mice were randomly divided into two groups,with one group receiving local injection of HUCMSCs suspension(HUCMSCs group)and the other receiving PBS(control group).Muscle tissues from the ischemic limb were collected at 3,7,and 14 d after surgery.HE staining and Masson staining were performed to observe morphological changes.LC-MS analysis was conducted in combination with KEGG database analysis to compare the metabolomic differences between the two groups.Results:HUCMSCs expressed high levels of CD105,CD90,and CD73,and low levels of HLA-DR,CD14,CD19,and CD34.Morphological observations revealed a significant improvement in muscle atrophy and fibrosis in the HUCMSCs group compared to the control group at 7 and 14 d after surgery.Non-targeted metabolomic analysis identified 687 metabolites in the gastrocnemius muscle samples,with lipids representing the largest proportion(34.088%).Differential metabolites included 37 in negative ion mode(25 upregulated,12 downregulated)and 17 in positive ion mode(11 upregulated,6 downr

关 键 词：外周血管疾病 间质干细胞 神经酰胺类 代谢组学 

分 类 号：R543[医药卫生—心血管疾病]