脂肪干细胞来源外泌体在组织损伤修复中的蛋白组学分析

文题释义：非标记定量蛋白质谱技术(label freequantification，LFQ)：即利用液质联用技术对蛋白质酶解肽段进行质谱分析，该技术优点是免去了高昂的稳定同位素标签做内标，大量样本检测时只需对蛋白质谱数据进行分析对比，检测样本中相应肽段的信号强度，对其相应的蛋白质进行相对定量分析。外泌体：为新兴的生物学标记物，目前广泛应用于肿瘤、免疫等领域，可作为疾病诊断、治疗的指标，具有较高的诊断敏感度及准确性。外泌体存在于绝大多数体液环境中，具备强大的旁分泌功能，因此具有作为药物载体的潜能。背景：脂肪干细胞促进损伤修复的研究较多，但从脂肪干细胞来源外泌体角度出发、基于蛋白质组学及生物信息学技术对其进行研究尚属少见。目的：通过蛋白质组学及生物信息学技术，分析糖尿病下肢溃疡患者和健康成年人脂肪干细胞来源外泌体差异表达蛋白，以期筛选诊断及预后标志物。方法：试验组为2018年10月至2019年5月在空军军医大学第二附属医院整形烧伤科接受清创植皮的糖尿病下肢溃疡患者25例、对照组为同期接受吸脂术的健康成年人10名。制备脂肪干细胞、提取外泌体并进行鉴定，从两组中分别随机抽取3例进行蛋白组学研究：利用LFQ技术行蛋白质定量分析；使用clueGO(version 2.5.4)对差异表达蛋白行GO和Pathway分析；对差异蛋白进行了蛋白-蛋白间相互作用分析(数据库String，版本11.0，R软件clusterProfiler package，FDR< 0.25)。结果与结论：①共筛选出可定量差异表达蛋白231种，其中31种差异有显著性意义，试验组下调表达蛋白29种，上调表达蛋白2种；②差异蛋白与营养、代谢类疾病相关性高；差异表达蛋白所处的细胞位置集中在细胞外囊泡、血小板α颗粒；差异表达蛋白参与的生物学过程为凝血过程及脂质转运；差异蛋白的主要分子功能与酶抑制剂活性、肝素结合能力有关；③PPI和KEGG通路分析显示，试验组表达下调显著的ITGA2B、ITGB3蛋白与病程密切相关，有望成为应用干细胞治疗糖尿病性下肢溃疡的新靶点。ORCID: 0000-0002-9022-5312(李学拥)；0000-0001-7327-2582(杨雯娴)中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Proteomics analysis of exosomes from adipose-derived stem cells in skin damage repair

BACKGROUND: There are lots of researches about using adipose-derived stem cells to promote damage repair, but the studies from the perspective of exosome from adipose-derived stem cells based on proteomics and bioinformatics technologies are rarely reported. OBJECTIVE: To analyze the differentially expressed proteins of exosome from adipose-derived stem cells between diabetic patients with leg ulcers and healthy adults through proteomics and bioinformatics technologies, so as to screen out the diagnostic and prognostic landmarks. METHODS: Adipose-derived stem cells were obtained from 25 diabetic patients(trial group) with leg ulcers who accepted debridement skin grafting at the Department of Plastic and Burn, Second Affiliated Hospital of Air Force Military Medical University from October 2018 to May 2019 and 10 healthy adults who concurrently accepted liposuction(control group). Exosomes from adipose-derived stem cells were extracted for identification. Three cases were randomly selected from each group for proteomics analysis. LFQ technology was used to conduct quantitative analysis. GO and Pathway analysis for the differentially expressed proteins were performed using clueGO(version 2.5.4) was used to do. The transactional analysis between proteins was conducted(String database, version 11.0, R software clusterProfiler package, FDR < 0.25). RESULTS AND CONCLUSION:(1) Totally 231 quantitative differentially expressed proteins were screened out, 31 kinds of which had significant difference. There were 29 down-regulated proteins and 2 up-regulated proteins in the trial group.(2) Differential proteins had a high correlation with nutrition and metabolic diseases. Differentially expressed proteins were mostly expressed in extracellular vesicles and platelet alpha granules and were mainly involved in two biological processes-coagulation process and lipid transport. The main molecular functions of differential proteins were related to enzyme inhibitor activity and heparin binding capacity.(3) The pathway analysis of PPI and KEGG showed that the significantly down-regulated ITGA2 B and ITGB3 proteins were closely related to the course of the disease in the trial group, both of which are expected to be the new targets in stem cell treatment for diabetes with leg ulcers.