小鼠部分肝切除后肝脏再生机制初探  

A preliminary investigation of the regenerative mechanism of liver regeneration after partial hepatectomy in mice

作　　者：鲁玫昕 张铭铭 赵玲萍 曾泉 习佳飞 裴雪涛 王海洋 岳文 LU Mei-xin;ZHANG Ming-ming;ZHAO Ling-ping;ZENG Quan;XI Jia-fei;PEI Xue-tao;WANG Hai-yang;YUE Wen(Stem Cell and Regenerative Medicine Lab,Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;South China Research Center for Stem Cell&Regenerative Medicine,SCIB,Guangzhou 510005,China)

机构地区：[1]军事科学院军事医学研究院卫生勤务与血液研究所军事再生医学研究室,北京100850 [2]华南生物医药研究院华南干细胞与再生医学研究中心,广州510005

出　　处：《军事医学》2022年第9期659-667,共9页Military Medical Sciences

基　　金：国家重点研发计划(2017YFA0103100,2017YFA0103103,2017YFA0103104);国家自然科学基金青年科学基金项目(8210114759)。

摘　　要：目的构建小鼠部分肝切除术(PH)后肝脏再生模型,初步探究肝脏再生的启动机制。方法对小鼠行PH手术,分别于术后12、24、36、48、72 h获取肝组织样本,同时设假手术组为对照,对肝组织进行病理、凋亡、BrdU掺入、增殖相关基因表达等检测;对实验组与对照组肝组织的单细胞转录组数据进行生物信息学分析,评估上皮细胞的细胞周期分布,以明确肝脏再生启动的时间;同时解析肝星状细胞(HSC)在再生不同阶段的分泌谱,筛选可能在肝脏再生启动中发挥功能的分泌因子;在体外进行细胞共培养实验,通过实时定量PCR、免疫荧光染色等方法检测HSC对肝细胞增殖的影响。结果成功构建小鼠2/3 PH模型,BrdU掺入检测和实时定量PCR结果显示,小鼠肝脏在PH后的早期基本不增殖,术后36~48 h达到增殖高峰;单细胞转录组分析同样表明大量上皮细胞在PH后48 h进入细胞周期S期;建立了HSC在再生不同阶段的分泌基因表达谱,筛选获得了包括Fgf9等在内的可能参与肝脏再生启动的候选分泌因子集合;进一步体外共培养实验表明人肝星状细胞系LX-2能够促进人肝细胞系THLE-2增殖。结论在小鼠PH后肝脏再生早期,微环境中的HSC分泌了多种促进细胞增殖的因子,且体外培养的HSC能够促进肝细胞增殖,提示HSC参与肝脏再生的启动并可能发挥正向调控作用。Objective To investigate the initiation mechanism of liver regeneration by establishing a mouse model of liver regeneration after partial hepatectomy(PH).Methods PH surgeries were performed on mice.The tissue samples were collected at 12,24,36,48 and 72 h post operation,and a sham-operated group was set up as a control.The pathological changes,apoptotic signals,BrdU incorporation,and expressions of proliferation-related genes were detected in the regenerating liver tissues.To find out about the time point of initiation of regeneration,bioinformatic analysis of the single-cell transcriptome data from the above liver tissues was performed and the cell cycle distribution of epithelial cells was assessed.Moreover,a secretion profile of hepatic stellate cells(HSCs)during regeneration was analyzed to investigate the regeneration-related secreted factors.A co-culture assay was performed in vitro,and the effect of HSCs on hepatocytes proliferation was detected by quantitative real-time PCR and immunofluorescence staining.Results A mouse PH model was constructed.The BrdU incorporation levels and the quantitative real-time PCR results showed that the mouse liver rarely proliferated at the early stage(within 24 h)after PH,but peaked at 36-48 h after PH.Transcriptome analysis at the single cell level showed that a large number of epithelial cells entered the S phase at 48 h after PH;The secretion profile obtained from HSCs at the early stage of regeneration revealed a spectrum of secretory factors including Fgf9,which was possibly involved in the initiation of liver regeneration.In vitro co-culture assays showed that LX-2 cells could promote the proliferation of THLE-2 cells.Conclusion During the early stage of liver regeneration after PH,HSCs may possibly secrete a variety of factors with pro-proliferation functions.Also,the HSCs cultured in vitro are able to promote the proliferation of hepatocytes.HSCs might participate in the initiation of liver regeneration and play a positive regulatory role.

关 键 词：部分肝切除术 肝脏再生 肝星状细胞 肝细胞增殖 

分 类 号：R34[医药卫生—基础医学] Q28[生物学—细胞生物学]