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

作　　者：安然 任昊桢[2] 管文贤[1] AN Ran;REN Haozhen;GUAN Wenxian(Department of Gastric and Hernia Surgery,Nanjing Drum Tower Hospital,the Affiliated Hospital of Medical School,Nanjing University,Nanjing,Jiangsu 210008,China;bDivision of Hepatobiliary and Liver Transplantation Surgery,Nanjing Drum Tower Hospital,the Affiliated Hospital of Medical School,Nanjing University,Nanjing,Jiangsu 210008,China)

机构地区：[1]南京大学医学院附属鼓楼医院胃与疝外科,江苏南京210008 [2]南京大学医学院附属鼓楼医院肝胆与肝移植外科,江苏南京210008

出　　处：《肝胆胰外科杂志》2025年第1期18-25,共8页Journal of Hepatopancreatobiliary Surgery

基　　金：国家自然科学基金项目(82270646)。

摘　　要：目的探讨过量肿瘤坏死因子α(TNF-α)对肝细胞增殖能力的影响以及间充质干细胞(MSCs)促进肝细胞增殖的具体机制。方法体外使用过量TNF-α作用于小鼠肝细胞系AML12并使用MSCs治疗。转录组测序分析阴性对照(NC)组和TNF-α组AML12细胞的差异表达基因(DEGs)以及基因功能富集和增殖通路的变化。使用公共数据库Gene Transcription Regulation Database(GTRD)预测转录因子(TF)并与DEGs交集,进一步分析调控增殖信号通路的关键因子。利用染色质免疫共沉淀(ChIP)、荧光素酶报告基因实验、蛋白免疫印迹(WB)、实时荧光定量聚合酶链式反应(qRT-PCR)以及CCK-8检测目标TF及其调控的下游靶基因和信号通路对肝细胞增殖的影响。结果转录组分析提示TNF-α组肝细胞增殖受限,经典的Wnt信号通路被显著负调控,其中β-catenin的转录受限,下游通路被抑制。通过GTRD预测β-catenin的TF并于DEGs交集发现,广泛调控细胞存活、增殖、迁移等生物学行为的TF特异蛋白1(SP1)的转录也受到抑制。慢病毒转染敲低AML12细胞SP1的表达后WB和qRT-PCR结果显示β-catenin及其下游增殖相关分子的蛋白和mRNA水平降低;ChIP-qRT-PCR和荧光素酶报告实验表明处理后SP1参与调控β-catenin的转录。对过量TNF-α处理后的AML12细胞进行WB、qRT-PCR、ChIP-qRT-PCR和荧光素酶报告实验发现,由SP1/β-catenin轴介导的细胞增殖被抑制。MSCs治疗提高了AML12细胞内SP1和β-catenin的蛋白含量,但被SP1抑制剂普卡霉素(MrA)显著抑制。qRT-PCR观察到相似的趋势,但MSCs治疗后AML12细胞内SP1的mRNA含量并未增加,提示MSCs并非通过促进SP1的转录发挥治疗作用。鉴于MSCs传递生物活性物质的功能,使用短发夹敲低SP1表达的MSCs进行治疗,结果表明,敲低SP1导致MSCs的上述治疗作用被显著抑制。结论过量TNF-α导致肝细胞内Wnt信号通路介导的增殖被抑制,MSCs通过传递SP1上调肝细胞β-catenin的表�Objective To explore the impact of excessive tumor necrosis factorα(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-αin 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-αgroup,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-αgroup,with significant negative regulation observed in the canonical Wnt signaling pathway,including restricted transcription ofβ-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 theβ-catenin promoter in the TNF-αgroup.WB and qRT-PCR results indicated that the protein and mRNA levels ofβ-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 ofβ-catenin transcription after treatment.WB,qRT-PCR,ChIP-qRT-PCR,and luciferase reporter assays showed that cell

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 

分 类 号：R575.3[医药卫生—消化系统]