体外连续传代对人脐带来源间充质干细胞转录组的影响  

Influence of continuous passaging in vitro on human umbilical cord-derived mesenchymal stem cell

作　　者：金琳琳 鲁安妮 陈晓莉 魏璇 段永娟 胡甜园 张英驰 JIN Linlin;LU Anni;CHEN Xiaoli;WEI Xuan;DUAN Yongjuan;HU Tianyuan;ZHANG Yingchi(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,TianjinInstitutes of Health Science,Tianjin300020,China)

机构地区：[1]中国医学科学院/北京协和医学院血液病医院(中国医学科学院血液学研究所)血液与健康全国重点实验室/国家血液系统疾病临床医学研究中心/细胞生态海河实验室天津医学健康研究院,天津300020

出　　处：《中华实用诊断与治疗杂志》2024年第4期342-347,共6页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家重点研发计划(2021YFA1101603,2019YFA0110803)。

摘　　要：目的分析不同代次人脐带来源间充质干细胞(hUC-MSC)转录组差异,探讨体外连续传代对hUC-MSC转录组的影响。方法从无菌脐带样本中分离脐带华通胶组织于细胞培养瓶中培养,培养1周时成纤维样细胞从组织块周围爬出,消化传代后获得低代次(第3代,P3)和高代次(第15代,P15)hUC-MSC。取P3和P15hUC-MSC细胞,行免疫表型和三系分化潜能鉴定;提取细胞RNA,构建测序文库进行转录组测序,获得P3、P15hUC-MSC转录组基因表达矩阵,以校正后P<0.05且|log2FC|≥2筛选差异表达基因,并进行基因本体(GO)和京都基因与基因组百科全书(KEGG)通路富集分析。为验证测序结果,采用实时荧光定量PCR法检测P3、P15hUC-MSC肿瘤坏死因子受体超家族成员11B(TNFRSF11B)mRNA相对表达量,采用ELISA法检测P3、P15hUC-MSC上清液TNFRSF11B蛋白水平。结果P3、P15hUC-MSC均表达CD73、CD90、CD105,均不表达CD14、CD34、CD45、HLA-DR;均可向成脂、成骨和成软骨细胞分化。P3、P15hUC-MSC共比对60656个基因表达情况,筛选出497个差异表达基因。与P3hUC-MSC比较,P15hUC-MSC上调基因有335个,包括TNFRSF11B、FGFR2、CCND2、WT1等;下调基因有162个,包括NFE2、IL-7R、C1QL4等。GO富集分析结果显示,差异表达基因共富集于204条GO条目,其中183条为生物过程,19条为细胞组分,2条为分子功能,主要富集于系统和器官发育、蛋白质磷酸化、细胞黏附等生物过程;细胞组分分析显示,差异表达蛋白主要富集于细胞表面及质膜等部位。KEGG通路富集分析结果显示,差异表达基因主要富集于PI3K-Akt、RAP1信号通路、轴突导向、细胞外基质受体相互作用和癌症中蛋白多糖增加等相关通路。P15hUC-MSC TNFRSF11B mRNA相对表达量(4.484±0.018)及上清液TNFRSF11B蛋白水平[(118.925±0.475)μg/L]均高于P3hUC-MSC[1.000±0.021、(22.046±0.116)μg/L](t=49.490,P<0.001;t=343.300,P<0.001)。结论hUC-MSC体外连续传代导致转录组发生变化,异�Objective To analyze the transcriptome differences between different generations of human umbilical cord-derived mesenchymal stem cell(hUC-MSC),and to investigate the effects of continuous passaging in vitro on the hUC-MSC transcriptome.Methods The Wharton's jelly was isolated from the procured umbilical cord and cultured in cell culture bottle.Fibroblast-like cells emerged from the tissue block after culture for 1 week,and low generation(P3)and high generation(P15)of hUC-MSC were obtained after digestion and passaging.First,immunophenotype and trilineage differentiation potential were identified in P3 and P15 hUC-MSCs and their RNAs were extracted to construct sequencing libraries for transcriptome sequencing.The transcriptome gene expression matrix of P3 and P15 hUC-MSCs were obtained.This matrix was screened for P3 and P15 hUC-MSCs differentially expressed genes(DEGs)with adjusted P<0.05 and|10g2FC|≥2,and analyzed for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).To verify the sequencing results,real-time fluorescence quantitative PCR was used to detect the relative expressions of tumor necrosis factor receptor superfamily member,tumor necrosis factor receptor superfamily member 11B(TNFRSF11B)mRNA of P3 and P15 hUC-MSCs.The levels of TNFRSF11B proteins in supernatant of P3 and P15 hUC-MSCs were detected by ELISA.Results P3 and P15 hUC-MSCs all expressed CD73,CD90 and CD105,but did not express CD14,CD34,CD45 and HLA-DR.All were differentiated into adipogenic,osteogenic and chondrogenic cells.The expressions of 60656 genes were compared,and 497 DEGs were obtained.Compared with P3 hUC-MSC,335 genes were up-regulated such as TNFRSF11B,FGFR2,CCND2 and WT1,and 162 genes were down-regulated such as NFE2,IL-7R and C1QL4 in P15 hUC-MSC.GO enrichment analysis revealed that DEGs were enriched in 204 GO entries,including 183 biological processes,19 cellular components,and 2 molecular functions,which were mainly enriched in biological processes such as system and organ development,protein phosphoryla

关 键 词：人脐带来源间充质干细胞 体外连续传代 转录组测序 肿瘤坏死因子受体超家族成员11B 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]