肝胆胰外科杂志2025，Vol.37Issue(1)：18-25,8.DOI:10.11952/j.issn.1007-1954.2025.01.004
间充质干细胞促进肝细胞增殖治疗急性肝衰竭的体外实验研究
Mesenchymal stem cells promote hepatocyte proliferation in the treatment of acute liver failure:An in vitro study
摘要
Abstract
Objective To explore the impact of excessive tumor necrosis factor α(TNF-α)on hepatocyte proliferation and to examine the specific mechanisms through which mesenchymal stem cells(MSCs)promote hepatocyte proliferation.Methods The AML12 cells were exposed to excess TNF-α in vitro and subsequently treated with MSCs.Transcriptome sequencing was employed to analyze the differentially expressed genes(DEGs)and gene enrichment in AML12 cells between the negative control(NC)group and the TNF-α group,to elucidate alterations in proliferation pathways.Gene Transcription Regulation Database(GTRD)was utilized to predict transcription factors,which were then intersected with DEGs to further elucidate key factors regulating proliferation signaling pathways.The impact of target transcription factors and their downstream target genes and signaling pathways on hepatocyte proliferation were analyzed by chromatin immunoprecipitation(ChIP),luciferase reporter assays,Western blotting(WB),quantitative real-time polymerase chain reaction(qRT-PCR)and Cell Counting Kit-8(CCK-8)assays.Results Transcriptome analysis revealed that hepatocyte proliferation was suppressed in the TNF-α group,with significant negative regulation observed in the canonical Wnt signaling pathway,including restricted transcription of β-catenin and inhibition of downstream pathways.Prediction from GTRD identified specificity protein 1(SP1),a transcription factor involved in regulating cell survival,proliferation,and migration,was inhibited.Further experiments demonstrated decreased binding ability of SP1 to the β-catenin promoter in the TNF-α group.WB and qRT-PCR results indicated that the protein and mRNA levels of β-catenin and its downstream proliferation-related molecules were reduced in AML12 cells after lentiviral transfection inhibited SP1 expression.ChIP-qRT-PCR and luciferase reporter assays demonstrated that SP1 was involved in the regulation of β-catenin transcription after treatment.WB,qRT-PCR,ChIP-qRT-PCR,and luciferase reporter assays showed that cell proliferation mediated by the SP1/β-catenin axis was inhibited in AML12 cells after excessive TNF-α treatment.Treatment with MSCs increased the protein levels of SP1 and β-catenin in AML12 cells,but this effect was significantly inhibited by the SP1 inhibitor Mithramycin A(MrA).Similar trends were observed in the qRT-PCR results,but the mRNA level of SP1 in AML12 cells did not increase after MSC treatment,suggesting that MSCs did not exert their therapeutic effects by promoting SP1 transcription in AML12 cells.Given the role of MSCs in delivering bioactive substances,MSCs with SP1 expression knocked down using short hairpins were used for treatment.The results showed that the therapeutic effects of MSCs were significantly inhibited by SP1 knockdown.Conclusion Excessive TNF-α leads to the inhibition of cell proliferation mediated by the Wnt signaling pathway.MSCs promote hepatocyte proliferation by upregulating β-catenin expression through SP1 delivery.关键词
间充质干细胞/肿瘤坏死因子α/转录因子/特异蛋白1/急性肝功能衰竭/肝细胞增殖/小鼠Key words
mesenchymal stem cell/tumor necrosis factor α/transcription factor/specificity protein 1/acute liver failure/hepatocyte proliferation/mice分类
临床医学引用本文复制引用
安然,任昊桢,管文贤..间充质干细胞促进肝细胞增殖治疗急性肝衰竭的体外实验研究[J].肝胆胰外科杂志,2025,37(1):18-25,8.基金项目
国家自然科学基金项目(82270646). （82270646）
