体外连续传代对人脐带间充质干细胞分泌组蛋白表型的影响

Effects of in vitro continuous passaging on secretory proteins of human umbilical cord-derived mesenchymal stem cells

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-1蛋白表达量均高于P15代hUC-MSCs培养上清液(P<0.05),IL-6、IL-11、IL-13、IL-17、M-CSF、TNFR2、MIP-1β、EGFR、GDNF、Ⅰ-309、GDF-15及MCP-1蛋白表达量均高于P10代hUC-MSCs培养上清液(P<0.05),IL-6、IL-11、IL-13、M-CSF、TNFR2、MIP-1β、EGFR、GDNF、Ⅰ-309蛋白表达量均高于P6代hUC-MSCs培养上清液(P<0.05)。P6代hUC-MSCs培养上清液中IL-6、IL-11、IL-13、GM-CSF、TNFR2、Eotaxin-1、EGFR、SCFR、GDNF和Ⅰ-309蛋白表达量均高于P15代hUC-MSCs培养上清液(P<0.05),TNFR2、GDNF蛋白表达量均高于P10代hUC-MSCs培养上清液(P<0.05)。P10代hUC-MSCs培养上清液中IL-6、IL-11、IL-13、GM-CSF、GDNF和Ⅰ-309蛋白表达量均高于P15代hUC-MSCs培养上清液(P<0.05)。(4)GO富集分析显示,15种差异表达蛋白主要在信号受体激活剂的活性、细胞因子介导的信号通路、细胞迁移的正向调节、生长因子活性等条目中富集。(5)KEGG通路富集分析结果显示,15种差异表达蛋白高富集于细胞因子受体的相互作用、病毒蛋白与细胞因子和细胞因子受体的相互作用、类风湿性关节炎以及IL-17和JAK-STAT信号通路等。结论 体外连续传代可导致hUC-MSCs分泌组蛋白表型变化,表现为高代次hUC-MSCs生长、分化、修复、免疫调节和趋化能力减弱,促炎和衰老表型增加。

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 there were no significant differences in the expressions of IL-1αand IL-7(P>0.05).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 supernatant of P4hUC-MSCs than those in the supernatant of P15hUC-MSCs(P<0.05).The expressions of IL-6,IL-11,IL-13,IL-17,M-CSF,TNFR2,MIP-1β,EGFR,GDNF,Ⅰ-309,GDF-15and MCP-1proteins were higher in the supernatant of P4hUC-MSCs than those in the supernatant of P10hUC-MSCs(P<0.05).The expressions of IL-6,IL-11,IL-13,M-CSF,TNFR2,MIP-1β,EGFR,GDNF andⅠ-309 proteins were higher in the supernatant of P4 hUC-MSCs than those in the supernatant of P6hUC-MSCs(P<0.05).The expressions of IL-6,IL-11,IL-13,GM-CSF,TNFR2,Eotaxin-1,EGFR,SCFR,GDNF andⅠ-309proteins were higher in the supernatant of P6hUC-MSCs than those in the supernatant of P15hUC-MSCs(P<0.05),and the expressions of TNFR2and GDNF proteins were higher in the supernatant of P6hUC-MSCs than those in the supernatant of P10hUC-MSCs(P<0.05).The expressions of IL-6,IL-11,IL-13,GM-CSF,GDNF andⅠ-309proteins were higher in the supernatant of P10hUC-MSCs than those in the supernatant of P15hUC-MSCs(P<0.05).(4)GO enrichment analysis showed that the 15differentially expressed proteins were mainly enriched in signaling receptor activator activity,cytokine-mediated signaling pathway,positive regulation of cell migration and growth factor activity,etc.(5)KEGG pathway enrichment analysis showed that the 15differentially expressed proteins were highly enriched in pathways such as cytokine-cytokine receptor interaction,viral protein interaction with cytokine and cytokine receptor,rheumatoid arthritis,and IL-17and JAK-STAT signaling pathways,etc.Conclusion Continuous passaging of hUC-MSCs in vitro could result in phenotypic changes of the secretory proteins,presenting as diminished capacities of growth,differentiation,repair,immunoregulation and chemotaxis as well as increased pro-inflammatory and senescent phenotypes in higher passage of hUC-MSCs.