摘要
目的 应用生信分析技术分析不同代次人脐带来源间充质干细胞(human umbilical cord derived mesenchymal stem cells, hUC-MSCs)分泌组蛋白表型差异,探讨体外连续传代对hUC-MSCs分泌组蛋白表型的影响。方法 取第4、6、10、15代(简写为P4、P6、P10、P15)hUC-MSCs的培养上清液,应用高通量人生长因子芯片(QAH-GF-1)、高通量人炎性因子芯片(QAH-INF-3)试剂盒检测80种细胞因子蛋白表达,微阵列数据分析软件Q-Analyzer自动计算出蛋白表达量。将检测浓度最佳置信度占比为50%~100%的蛋白浓度视为质控合格的可靠值,筛选出质控合格的蛋白,应用TBtools 1.105软件绘制层次聚类热图,分析不同代次hUC-MSCs培养上清液中蛋白的表达趋势,记录表达量随体外传代次数增多而增高或降低的蛋白,应用GraphPad Prism 8.0.1软件进行单因素方差分析,以P<0.05为标准筛选出差异表达蛋白,应用在线工具Metascape和微生信进行基因本体(gene ontology, GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)富集分析。结果 (1)人炎性因子芯片(QAH-INF-3)中有25种蛋白、人生长因子芯片(QAH-GF-1)中有14种蛋白质控合格。(2)质控合格蛋白的聚类分析结果显示,白细胞介素(interleukin, IL)-6、IL-11、IL-13、IL-17、M-CSF、GM-CSF、TNFR2、Eotaxin-1、MIP-1β、EGFR、SCFR、GDNF、Ⅰ-309、GDF-15、MCP-1及IL-1α共16种蛋白表达随体外扩增代次增加呈逐代下调趋势,IL-7表达随体外扩增代次增加呈逐代上调趋势。(3)P4、P6、P10和P15代hUC-MSCs培养上清液中IL-6、IL-11、IL-13、IL-17、M-CSF、GM-CSF、TNFR2、Eotaxin-1、MIP-1β、EGFR、SCFR、GDNF、Ⅰ-309、GDF-15及MCP-1蛋白表达量比较差异均有统计学意义(P<0.05),IL-1α、IL-7蛋白表达量比较差异均无统计学意义(P>0.05)。P4代hUC-MSCs培养上清液中IL-6、IL-11、IL-13、IL-17、M-CSF、GM-CSF、TNFR2、Eotaxin-1、MIP-1β、EGFR、SCFR、GDNF、Ⅰ-309、GDF-15及MCP
Objective To analyze the difference of the phenotype of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)secretory proteins with bioinformatic analysis technique,and to investigate the effect of continuous in vitro passaging on the phenotype of hUC-MSCs secretory proteins.Methods The cultural supernatants of the 4th,6th,10th and 15th passages(P4,P6,P10 and P15)of hUC-MSCs were obtained,and the expression levels of 80 proteins were measured by high-throughput antibody microarray quantitative techniques,including human growth factor array(QAH-GF-1)and human inflammatory factor array(QAH-INF-3)kits.The expressions of proteins were automatically calculated with microarray data analysis software Q-Analyzer.Concentrations of the proteins with the best confidence ratio of 50%to 100%were set as reliable values for quality control.TBtools software was used to draw a clustering heatmap for screening out the proteins that regularly increased or decreased with continuous passaging in vitro,and GraphPad Prism 8.0.1software was used to perform one-way ANOVA analysis in order to select differentially expressed proteins with P<0.05.The online tools such as Metascape and Bioinformatics were applied for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Results(1)There were 25proteins in QAH-INF-3and 14proteins in QAH-GF-1that were detected with satisfactory quality control results.(2)The clustering analysis of the quality control-eligible proteins showed that with the increase of in vitro amplification,the expression levels of 16proteins including interleukin(IL)-6,IL-11,IL-13,IL-17,M-CSF,GM-CSF,TNFR2,Eotaxin-1,MIP-1β,EGFR,SCFR,GDNF,Ⅰ-309,GDF-15,MCP-1and IL-1αexhibited a gradually down-regulated trend,while that of IL-7was gradually up-regulated.(3)There were significant differences in the expressions of IL-6,IL-11,IL-13,IL-17,M-CSF,GM-CSF,TNFR2,Eotaxin-1,MIP-1β,EGFR,SCFR,GDNF,Ⅰ-309,GDF-15and MCP-1proteins in the cultural supernatants of P4,P6,P10and P15hUC-MSCs(P<0.05),and th
作者
魏璇
段永娟
吴沂璇
蔡玉丽
金琳琳
胡甜园
章婧嫽
张英驰
WEI Xuan;DUAN Yong-juan;WU Yi-xuan;CAI Yu-li;JIN Lin-lin;HU Tian-yuan;ZHANG Jing-liao;ZHANG Ying-chi(State Key Laboratory of Erperimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology and Blood Diseases Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Tianjin Institutes of Health Science,Tianjin 300020,China)
出处
《中华实用诊断与治疗杂志》
2023年第5期448-454,共7页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家重点研发计划(2019YFA0110803)
国家重点研发计划(2021YFA1101603)。
关键词
人脐带来源间充质干细胞
分泌组蛋白
连续传代
基因本体分析
京都基因与基因组百科全书富集分析
human umbilical cord-derived mesenchymal stem cells
secretory proteins
continuous passaging
gene ontology analysis
Kyoto Encyclopedia of Genes and Genomes analysis
