摘要
目的观察人脐带间充质干细胞(hUCMSC)连续传代过程中基因组甲基化动态变化,探讨体外连续传代对hUCMSC基因组甲基化的影响。方法从无菌脐带样本中获取脐带华通胶组织,在细胞培养瓶中培养1周后可见hUCMSC(P0代次),经体外连续传代培养获得低代次(P3)、中代次(P6)和高代次(P15)hUCMSC。提取hUCMSC DNA,进行亚硫酸氢盐处理并纯化。构建DNA文库,并进行文库扩增、纯化,应用高通量基因组DNA甲基化检测技术,将850K甲基化芯片探针与DNA文库杂交,将杂交后的DNA片段通过XStain试剂标记后,采用iScan系统扫描获取IDAT文件得到P3、P6、P15 hUCMSC全基因组甲基化测序结果。应用R语言Champ.DMP包(以Δβ>0.2为高甲基化,Δβ<-0.2为低甲基化)筛选差异CpG岛甲基化位点。采用实时荧光定量PCR法和Western blot法分别检测P3、P15 hUCMSC人表皮生长因子受体(EGFR)、E2F转录因子1(E2F1)、周期蛋白依赖性激酶抑制剂2A(CDKN2A)mRNA和蛋白相对表达量。应用R语言Champ.DMR包识别差异甲基化区域,采用Champ.Annotate包整合EPIC阵列注释基因定位特征,采用Champ.GSEA包对差异甲基化基因进行基因本体(GO)富集分析,采用ggplot2包生成差异甲基化基因的热图可视化报告及注释。结果P3与P6 hUCMSC存在889个CpG岛差异甲基化位点,涉及7个基因,其中1个为高甲基化位点,6个为低甲基化位点;与P3相比,P6 hUCMSC位于PKNOX1基因TSS1500 shore甲基化水平显著上调。P6与P15 hUCMSC存在6783个CpG岛差异甲基化位点,涉及14个基因,其中7个为高甲基化位点,7个为低甲基化位点;与P6相比,P15 hUCMSC位于E2F1基因ExonBnd opensea甲基化水平下调,位于PRDM2基因5′UTR shore甲基化水平上调。P3与P15 hUCMSC存在18327个差异甲基化位点,涉及10个基因,其中7个为高甲基化位点,3个为低甲基化位点;与P3相比,P15 hUCMSC位于EGFR基因IGR opensea甲基化水平下调,位于CDKN2A基因Body shelf甲基化水平上调。P15 hUCMSC EGFR mRNA和蛋白(1.14±0.03、1.73±0.19)、E2F1蛋白(1.67±0.15)相对表达量均高于P3 hUCMSC(1.00±0.07、0.78±0.02、0.70±0.03)(t=-3.118、-8.730、-10.910,P均<0.05),CDKN2A mRNA和蛋白相对表达量(0.76±0.06、0.79±0.09)均低于P3 hUCMSC(0.99±0.06、1.32±0.01)(t=4.908,P=0.008;t=20.710,P<0.001),E2F1 mRNA相对表达量(1.03±0.02)与P3 hUCMSC(0.99±0.03)比较差异无统计学意义(t=-1.689,P=0.168)。差异甲基化分析热图显示,P3与P6 hUCMSC差异甲基化位点主要集中在Body区域,其次为IGR区域;P3与P15 hUCMSC差异甲基化位点数量明显多于P3与P6 hUCMSC差异甲基化位点数量,且低甲基化位点差异数量多于高甲基化位点,IGR区域的差异甲基化位点数量最多,有7000多个且集中于IGR区域。GO富集分析结果显示,P3与P15 hUCMSC的差异甲基化基因主要参与分子功能、生物学过程、细胞组分,涉及细胞周期、DNA修复、细胞增殖、干细胞分化、凋亡和衰老等。结论hUCMSC长期体外扩增引发表观遗传失衡,驱动细胞周期相关基因异常甲基化,表现为细胞增殖倾向于失控与细胞干性衰退。
Objective To observe the temporal dynamics of DNA methylation patterns in human umbilical cord-derived mesenchymal stem cells(hUCMSCs)during prolonged in vitro serial passaging,and to elucidate the impact of in vitro serial passaging on hUCMSC genome methylation.Methods Human umbilical cord Wharton's jelly-derived tissue was aseptically dissected from sterile umbilical cord samples.Primary hUCMSCs(P0)were established after 1-week culture in cell culture flask,with subsequent in vitro serial passaging to obtain early-(P3),mid-(P6),and late-passage(P15)hUCMSCs.hUCMSC DNA was extracted,bisulfite-converted,and purified.A DNA library was constructed,amplified,and purified.High-throughput genomic DNA methylation detection technology was applied,hybridizing the 850K methylation array probes with the DNA library.After hybridization,the DNA fragments were labeled with XStain reagent and scanned on the iScan system to obtain IDAT files,resulting in whole-genome methylation sequencing data for P3,P6 and P15 hUCMSCs.Differentially methylated CpG sites were screened using the R package Champ.DMP(with△β>0.2 defined as hypermethylation and△β<-0.2 defined as hypomethylation).The relative mRNA and protein expressions of human epidermal growth factor receptor(EGFR),E2F transcription factor 1(E2F1),and cyclin-dependent kinase inhibitor 2A(CDKN2A)in P3 and P15 hUCMSCs were detected using real-time quantitative PCR and Western blot,respectively.Differentially methylated regions were identified using the R package Champ.DMR.The EPIC array annotation gene location features were integrated using the Champ.Annotate package.Gene Ontology(GO)enrichment analysis was performed on differentially methylated genes using the Champ.GSEA package.Heatmaps and annotations visualizing the differentially methylated genes were generated using the ggplot2 package.Results Totally 889 differentially methylated CpG sites(DMCs)were identified in P3 and P6 hUCMSCs,involving 7 genes(1 hypermethylated,6 hypomethylated).P6 hUCMSCs exhibited significant hypermethylation at the TSS1500 shore region of PKNOX1 compared to P3 hUCMSCs(P<0.05).Totally 6783 DMCs were identified in P6 and P15 hUCMSCs,involving 14 genes(7 hypermethylated,7 hypomethylated).P15 hUCMSCs exhibited significantly hypomethylation at ExonBnd opensea region of E2F1,and hypermethylation at 5'UTR shore region of PRDM2 compared to P6 hUCMSCs.Totally 18327 DMCs were identified in P3 and P15 hUCMSCs,involving 10 genes(7 hypermethylated,3 hypomethylated).P15 hUCMSCs exhibited hypomethylation at the IGR opensea of EGFR and hypermethylation at the Body shelf region of CDKN2A compared to P3 hUCMSCs.The relative expressions of P15 hUCMSC EGFR mRNA and protein(1.14±0.03,1.73±0.19)and E2F1 protein(1.67±0.15)were higher than those of P3 hUCMSC(1.00±0.07,0.78±0.02,0.70±0.03)(t=-3.118,-8.730,-10.910;all P values<0.05).The relative expressions of P15 hUCMSC CDKN2A mRNA and protein(0.76±0.06,0.79±0.09)were lower than those of P3 hUCMSC(0.99±0.06,1.32±0.01)(t=4.908,P=0.008;t=20.710,P<0.001).The relative expression of P15 hUCMSC E2F1 mRNA showed no significant difference compared with that of P3 hUCMSC(1.03±0.02 vs 0.99±0.03)(t=-1.689,P=0.168).DMCs heatmaps demonstrated that P3 and P6 hUCMSC DMCs predominantly localized in Body region,followed by IGR region.The P3 and P15 hUCMSCs comparison showed marked expansion of DMCs compared to P3 and P6 hUCMSCs,particularly in IGR region(>7000 sites),with more hypomethylation DMCs than hypermethylation DMCs.GO enrichment analysis revealed that differentially methylated genes between P3 and P15 hUCMSCs were primarily involved in molecular function,biological process,and cellular component categories,with specific involvement in cell cycle,DNA repair,cell proliferation,stem cell differentiation,apoptosis and senescence.Conclusion Long-term in vitro expansion of hUCMSCs induces epigenetic imbalance,and drives aberrant methylation of cell cycle-related genes,which manifests as a tendency towards uncontrolled cell proliferation and a decline in stem cell potency.
作者
郭珂辛
黄雨馨
徐海惠
胡梓骐
段永娟
曹红文
张英驰
GUO Kexin;HUANG Yuxin;XU Haihui;HU Ziqi;DUAN Yongjuan;CAO Hongwen;ZHANG Yingchi(National Key Laboratory of Blood and Health,Hematology Hospital,Peking Union Medical College,Chinese Academy of Medical Sciences(Institute of Hematology,Chinese Academy of Medical Sciences)National Clinical Medical Research Center for Diseases of the Blood System Haihe Laboratory of Cellular Ecology Tianjin Institute of Medicine and Health,Tianjin 300020,China;Department of Obstetrics,Baodi Hospital of Tianjin Medical University,Tianjin 301800,China)
出处
《中华实用诊断与治疗杂志》
2025年第6期481-489,共9页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家重点研发计划项目(2021YFA1101603)。
关键词
人脐带间充质干细胞
甲基化
CPG岛
细胞周期
DNA修复
human umbilical cord-derived mesenchymal stem cells
methylation
CpG islands
cell cycle
DNA repair
