摘要
目的基于转录组测序(RNA-Seq)技术探讨猪苓多糖改变人原代巨噬细胞生长形态的可能分子机制。方法通过提取粗多糖并进行分离和纯化得到猪苓多糖。Ficoll法分离新生儿脐带血来源的外周血单个核细胞(PBMC),并通过CD14磁珠分选及重组人巨噬细胞集落刺激因子(M-CSF)诱导人巨噬细胞,连续5 d观察100 ng/ml猪苓多糖对其生长形态的影响。实验组取巨噬细胞使用猪苓多糖干预48 h,对照组不进行处理,对两组细胞进行转录组测序筛选差异基因;将得到的测序结果进行差异基因表达的GO、KEGG功能注释及信号通路显著性富集分析。结果诱导巨噬细胞具有贴壁生长及伪足分化能力,贴壁后细胞呈梭形和多角形,伪足较细长。100 ng/ml猪苓多糖处理能显著影响巨噬细胞的生长状态,巨噬细胞伪足逐渐消失且细胞变圆,最终分化褪失。比较两组测序结果发现差异表达基因共13825个,其中上调基因7028个,下调基因6797个。差异基因的GO功能显著性富集分析显示,差异基因主要集中在细胞外基质(ECM)过程。差异基因的信号通路显著性富集分析结果主要集中在ECM合成与细胞黏附通路。结论通过RNA-Seq技术证实猪苓多糖影响巨噬细胞的生长状态的分子机制依赖于ECM相关基因及黏附蛋白合成相关基因实现,为进一步研究猪苓多糖对巨噬细胞自身生理功能的调节作用提供依据。
Objective To investigate the possible molecular mechanism underlying altered growth morphology of human primary-culture macrophages induced by Polyporus umbellatus polysaccharide(PP)based on transcriptome RNA sequencing(RNA-Seq)techniques.Methods PP were obtained by extraction and separation from crude polysaccharide.Ficoll method was used to isolate peripheral blood mononuclear cells(PBMCs)from neonatal umbilical cord blood.Human macrophages were generated through CD14 magnetic cell sorting and induction with recombinant human macrophage colony-stimulating factor(M-CSF).The effects of PP(100 ng/ml)on morphology were observed for 5 days.Macrophages in the experimental group were treated with PP for 48 h,while those in the control group were not.RNA-Seq was used to screen for differential genes in the two groups of cells.The results of sequencing were subjected to GO annotation analysis and KEGG pathway enrichment analysis of the differential gene expression.Results Macrophages generated by induction were capable of adherent growth and pseudopodia differentiation.The adherent cells were spindle-shaped or polygonal,with slender pseudopodia.PP(100 ng/ml)could significantly affect the growth of macrophages,leading to gradually diminishing pseudopodia,cell rounding,and finally regression of cell differentiation.Comparison of the sequencing in the two groups identified 13825 differentially expressed genes,of which 7028 genes were up-regulated and 6797 genes were down-regulated.GO function enrichment analysis showed that the differentially expressed genes were mainly concentrated in the extracellular matrix(ECM)process.Pathway enrichment analysis of differential genes showed that they were mainly concentrated in ECM synthesis and cell adhesion pathway.Conclusion Using RNA-Seq techniques,it is showed that the molecular mechanism by which PP affects the growth status of macrophages depends on extracellular matrix-related genes and adhesion protein synthesis-related genes.These provides a basis for further study on the reg
作者
何敏
常雅彬
曾丹宁
周果
董艺红
谭庆龙
He Min;Chang Yabin;Zeng Danning;Zhou Guo;Dong Yihong;Tan Qinglong(Center of Translational Medicine,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China)
出处
《中华生物医学工程杂志》
CAS
2021年第3期252-259,共8页
Chinese Journal of Biomedical Engineering
基金
中国博士后基金面上项目(2019M652862)。
关键词
猪苓多糖
巨噬细胞
细胞外基质
计算生物学
转录组测序
Polyporus umbellatus polysaccharide
Macrophages
Extracellular matrix
Computational biology
RNA-seq
