文献深度解析：通过单细胞转录组解析马洛里 - 登克小体相关肝纤维化发病过程中的 HGF/MET 信号轴

一、文献基础综述

（一）期刊、基础信息

期刊：Signal Transduction and Targeted Therapy（STTT，信号转导与靶向治疗） 2026 最新 IF=81.2

DOI：10.1038/s41392-026-027224

完整标题：Unraveling the HGF/MET axis in Mallory-Denk body pathogenesis associated with liver fibrosis through single-cell transcriptomics

（二）全文核心结论

1.利用单核转录组（snRNA-seq）首次解析马洛里 - 登克小体（MDB）纤维化肝脏单细胞图谱，鉴定两类 MDB 相关肝细胞 MAH（Hep4/Hep5）、4 种肝星状细胞（HSC）亚群，证实 MAH 与肝细胞癌（HCC）进展高度相关，Slc7a11 为肝癌不良预后标志物；

2.明确肝内巨噬细胞（KCs）- 活化肝星状细胞（aHSC）-MAH细胞通讯轴核心通路为 HGF/MET；aHSC、KCs 分泌 HGF 结合 MAH 细胞膜 MET 受体，激活 PI3K/AKT/NF-κB、STAT3 双重下游信号；

3.HGF/MET 通路持续激活上调 UbD，诱导促炎因子 TNFα 释放，形成 “HGF-TNFα” 正反馈环路，驱动 MDB 蛋白聚集体形成；同时 aHSC 受通路调控分泌 TGFβ1，加剧肝纤维化；

4.UbD 基因敲除（UbD?/?）小鼠模型可显著抑制 HGF/MET 信号、阻断 MDB 生成、减轻胶原沉积与肝纤维化；人 MDB 肝癌样本中 HGF/MET、UbD 通路显著高表达；

5.首次构建 DDC 诱导 3D MDB 肝类器官（MDO）体外模型，完整复刻 MDB 病理特征，证实 HGF/MET/UbD 轴是慢性肝病、肝纤维化、肝癌联合潜在治疗靶点。

（三）Absin 产品作用简述

本研究采用abs9516 小鼠肝脏类器官培养试剂盒搭建 3D MDB 类器官体外病理模型，替代传统二维细胞系还原肝脏细胞互作微环境，为通路功能验证提供类器官核心培养体系；依托该试剂盒实现类器官稳定传代、冻存复苏，支撑 MET 抑制剂药物干预实验的数据产出。

二、研究领域背景介绍

慢性肝病（酒精性肝炎、代谢相关脂肪性肝炎 MASH、肝纤维化、肝癌）进程中普遍存在 MDB 蛋白包涵体，MDB 是肝细胞角蛋白 K8/K18、p62 泛素蛋白异常聚集产物，直接关联肝细胞气球样变、炎症、纤维化恶变，但驱动 MDB 形成的肝内细胞交互网络、核心分子通路长期不清晰。

肝星状细胞 HSC 是肝纤维化胶原主要来源，静息 HSC 激活为 aHSC 后大量分泌 ECM、促纤维化因子；库普弗细胞 KCs 是肝脏固有巨噬细胞，二者与损伤肝细胞形成复杂旁分泌调控网络，但 MDB 发生时三者间配体 - 受体通讯机制缺乏单细胞分辨率证据。

HGF/MET 通路经典功能为肝细胞修复再生，但在 MDB 相关纤维化中的双向调控、上下游级联分子（UbD）未知；既往研究仅使用批量转录组，无法区分异质性肝细胞、HSC 亚群功能差异，且缺少可稳定传代的体外 MDB 病理模型限制机制验证。

基于上述空白，本研究依托 snRNA-seq 单细胞技术、DDC 小鼠体内模型、Absin 小鼠肝类器官试剂盒构建 3D 体外 MDB 体系，联合 UbD 敲除基因动物，完整解析 MDB 发生、纤维化进展的 HGF/MET/UbD 分子调控轴。

三、作者整体递进研究思路总结

全文遵循动物病理建模→单细胞图谱绘制→细胞互作筛选核心通路→3D 类器官体外验证→细胞分子机制解析→基因敲除体内回证→人临床样本转化验证完整 8 层转化逻辑链：

1. DDC 饮食诱导小鼠建立稳定 MDB 肝纤维化模型，设置正常 / 给药 / 撤药 / 复药四组，病理染色证实炎症、脂肪变性、胶原沉积、MDB 表型；
2. 四组肝脏组织开展 snRNA-seq，总计 35588 个细胞核转录组，分群鉴定 9 大类肝脏细胞，重新聚类细分肝细胞 6 亚群、HSC4 亚群，锁定 MDB 特异性 MAH（Hep4/Hep5）、活化 aHSC；
3. 配体 - 受体互作分析筛选出 KCs、aHSC 分泌 HGF 与 MAH 细胞膜 MET 为最强通讯配对，GSVA 证实 MAH 内 PI3K/AKT/NF-κB、STAT3 通路显著富集；
4. 使用 Absin abs9516 试剂盒构建 3D MDB 类器官（MDO），建立肝细胞 - 肝星状细胞共培养体系，体外验证 HGF 促纤维化、促 MDB 形成功能；
5. 肝癌细胞 Hepa1-6 体外 TNFα/IFNγ 诱导 MDB 表型，HGF 刺激、MET/PI3K 抑制剂阻断正反双向实验，明确下游 NF-κB、STAT3 介导 Ub 转录上调；
6. CRISPR 构建 UbD?/?敲除小鼠 DDC 纤维化模型，反向验证 UbD 缺失抑制 HGF/MET 通路、缓解 MDB 与纤维化；
7. 公共肝癌队列 TCGA-LIHC、多套小鼠 / 人肝疾病转录数据集验证 MAH 标志物（Slc7a11、K8、UbD、Mmp14）临床预后价值；
8. 人 MDB 阳性肝癌活检组织蛋白、免疫荧光检测，证实 HGF/MET/UbD 通路在人类肝病中临床相关性，提出靶向治疗策略。

四、分模块详细研究思路、实验结果、对应原文图片

模块 1：DDC 小鼠 MDB 纤维化模型构建 + 肝脏单细胞图谱绘制

1.研究逻辑：DDC 诱导建立体内 MDB 模型，多组病理染色确认纤维化、MDB 病变；snRNA-seq 解析全肝脏细胞异质性，明确疾病进程中细胞比例变化。

2.核心实验：H&E、Masson、油红 O、α-SMA/F4/80 免疫组化；ALT/AST/ALP 血清生化；snRNA-seq 建库、UMAP 聚类、细胞比例统计；差异基因热图、小提琴标记基因图。

3.关键实验结果：

• DDC 饲喂 / 复药组小鼠肝脏肿大，转氨酶升高，出现显著胆管反应、肝细胞气球样变、脂沉积、胶原纤维、MDB 包涵体；CCl?联合 DDC 可进一步加重纤维化；
• snRNA-seq 分群得到肝细胞、HSC、库普弗细胞等 9 大类细胞；DDD 处理后 KCs、HSC、胆管细胞占比显著上升，肝细胞比例下降；
• 细胞标记基因热图清晰区分各类细胞分群，证实单细胞分群可靠性。

Fig. 1 MDB pathogenesis is associated with liver fibrosis and cellular heterogeneity. a Liver fibrosis, lipid deposition, and macrophage activation (F4/80) were assessed in the four groups via H&E, Masson, Oil Red O, and α-SMA IHC staining. Brown pigment deposition (black arrow) and MDBs (yellow arrow) were evident in MDB-forming livers. Scale bar: 100 μm. Images were captured at ×20 magnification. Three mice per group were quantified, with 3 fields analyzed per mouse. b, c RT?qPCR analysis of fibrosis markers (Acta2, Cdh2, and Vim) and collagen genes (Col1a1, Col3a1, and Col4a1) in liver homogenates. d Representative Masson, α-SMA and H&E staining showing fibrosis and MDB formation (yellow arrows). Scale bar: 100 μm; ×20 magnification. Quantification was performed as in (a). e RT?qPCR analysis of fibrosis related genes. f UMAP plots of the single-cell atlas from four groups showing nine major cell types categorized by sample origin (top) and cell type identity (bottom). g Violin plots showing marker gene expression for the nine cell types. h Bar plots of cell type proportions across samples. i Heatmap of the top DEGs (Wilcoxon test) for each cell type. The data are presented as the means ± SEMs; n = 3 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001

模块 2：鉴定 MDB 特异性肝细胞亚群 MAH（Hep4/Hep5）及其肝癌预后关联

1.研究逻辑：肝细胞亚群重聚类，筛选高表达 MDB 标志物（K8/K18/Sqstm1/UbD）细胞亚群；分化潜能、拷贝数变异 CNV、公共肝癌队列生存分析关联肿瘤进展。

2.核心实验：肝细胞亚群 UMAP 重分群、比例统计；MDB 标志物小提琴图；CytoTRACE 分化潜能分析；InferCNV 基因组不稳定性分析；TCGA-LIHC 队列反卷积、KM 生存曲线；多公共数据集基因表达验证。

3.关键实验结果：

• Hep4、Hep5 在 DDC 处理组比例大幅扩增，高表达 K8、K18、p62、UbD，定义为 MAH；Hep4 富集肝癌、凋亡通路，Hep5 富集氧化磷酸化、神经变性通路；
• CytoTRACE 提示 Hep4 分化程度最低、恶性潜能最高；Hep4 存在 1/6 号染色体拷贝数扩增，基因组不稳定；
• TCGA 肝癌队列中 Hep4 比例越高总生存期越短；Slc7a11 高表达肝癌患者预后差，可作为独立不良预后标志物。

Fig. 2 Emergence and molecular signature of MDB-associated hepatocytes (MAHs). a UMAP plots showing six hepatocyte subclusters across the control, DDC-Fed, DDC-Withdrawn, and DDC-Refed groups. b Bar plots of hepatocyte subcluster proportions in each group. c Violin plots of MDB marker gene expression across clusters. d Expression patterns of K8, K18, Sqstm1, and UbD in Hep4 (top) and Hep5 (bottom) cells across groups. e, f RT?qPCR validation of the expression of selected genes in Hep4 and Hep5 cells from DDC-treated versus control livers. g Heatmap of the top 10 DEGs (Wilcoxon test) across hepatocyte clusters. h KEGG enrichment of DEGs in Hep4 (top) and Hep5 (bottom) cells; adjusted p < 0.05. The data are shown as the means ± SEMs (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3 The MAH subset is closely associated with HCC progression. a, b CytoTRACE analysis showing the differentiation states of hepatocyte subsets, ordered from mature (low values) to immature (high values). c, d CNV profiles and distribution scores inferred by InferCNV across hepatocyte subsets and endothelial cells. e Hepatocyte subset composition (Hep0–Hep5) in HCC patients (TCGA-LIHC). f Kaplan–Meier survival analysis of hepatocyte subsets. g Expression of K8, K18, Slc7a11, and UbD across liver disease stages in a public database. h Kaplan–Meier survival analysis of Slc7a11 expression in TCGA-LIHC cohort

模块 3：肝星状细胞 HSC 亚群异质性，aHSC 促纤维化标志物 Mmp14 临床验证

1.研究逻辑：HSC 重聚类区分静息 / 活化亚群；通路富集明确 aHSC 纤维化、EMT 功能；临床肝癌样本验证 Mmp14 预后价值。

2.核心实验：HSC 亚群 UMAP、标记基因小提琴图；KEGG/GSEA 通路富集；Mmp 家族气泡图；WB、qPCR 验证；人肝癌组织免疫印迹、GEPIA 生存分析。

3.关键实验结果：

• HSC 分为 4 个亚群，活化 aHSC 在 DDC 组显著增多，高表达 Mmp14、α-SMA，富集 ECM 黏附、EMT、PI3K 炎症通路；
• Mmp14 在 aHSC 特异性高表达，促进 HSC 向肌成纤维细胞转化；人肝癌肿瘤组织 Mmp14、α-SMA 上调，高 Mmp14 提示更差生存期。

Fig. 4 Single-cell analyses identify distinct HSC populations in MDB livers. a UMAP visualization showing aHSC, qHSC, Mmp14-Bmp2?, and Mmp14-Il7r? subpopulations in control, DDC-Fed, DDC-Withdrawn, and DDC-Refed livers. b Violin plots of marker genes for each subcluster. c Contribution of each group to the HSC subclusters. d KEGG circular plot of enriched pathways in aHSCs from DDC-Fed livers. e GSEA of EMT and myogenesis in aHSCs versus other clusters. f, g Violin plots and bubble maps of Mmp gene expression across HSC subsets. h RT?qPCR validation of Mmp14 and Mmp2 expression in control vs DDC-treated livers. i Western blot analysis of Mmp14 in DDC and control mice. j Western blot of MMP14 and α-SMA in paired tumor and nontumor tissues from HCC patients. k Kaplan–Meier survival analysis of LIHC patients stratified by Mmp14 expression (TCGA, GEPIA). The data are shown as the means ± SEMs (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

模块 4：细胞互作筛选 HGF/MET 核心通路 + Absin 3D MDB 类器官体外模型验证

1.研究逻辑：配体 - 受体互作筛选 KCs/aHSC 与 MAH 核心通讯分子；使用 Absin abs9516 小鼠肝类器官试剂盒构建 3D MDB 体外模型，共培养验证肝细胞 - HSC 旁分泌互作。

2.核心实验：细胞间配体受体热图、互作气泡图；巨噬 - 肝细胞共培养 HGF ELISA；Absin 试剂盒 3D 类器官培养、传代冻存；类器官与 JS-1 肝星状细胞直接 / 间接共培养；TGFβ1、Mmp14、HGF 分泌 ELISA。

3.Absin 产品关键实验步骤：采用 Absin abs9516 小鼠肝脏类器官培养试剂盒，消化 DDC 诱导 MDB 小鼠原代肝细胞，基质胶包埋构建 3D MDB 类器官；试剂盒配套完全培养基支持类器官稳定扩增、7-9 天传代，冻存复苏后仍保留 MDB 标志物表达；用于 MET 抑制剂药物干预、HSC 共培养功能实验。

4.关键实验结果：

• HGF-MET 为 MAH 与 KCs、aHSC 最强互作配对；MDB 肝细胞可激活巨噬细胞大量分泌 HGF；
• Absin 试剂盒构建的 3D MDB 类器官稳定表达 K8、p62、UbD；类器官分泌可溶性因子诱导 HSC 释放 TGFβ1、Mmp14，放大纤维化信号；
• DDC 小鼠、人 MDB 肝癌血清 HGF 浓度显著高于对照。

Fig. 5 Enrichment of the HGF/MET axis during MDB pathogenesis. a Heatmap of ligand–receptor interactions between hepatocytes and HSCs/KCs in DDC-Fed and DDC-Refed livers. b, c Dot plots of enriched ligand?receptor interactions between hepatocytes and KCs (b) or aHSCs (c). d Bar plot of receptor–ligand interactions between Hep4/Hep5 cells and the indicated cell types. e GSVA of enriched pathways in hepatocyte subclusters. f RT?qPCR and ELISA results showing HGF expression in Raw264.7 cells cocultured with MDB-forming Hepa1-6 cells. g Experimental schematic of mouse MDB organoid (MDO) cultures. h, i Representative images of DDC-induced MDOs across time points (h) and passages (i). Scale bar: 100 μm. j Western blot of K8, UbD and p62 in organoids from control vs DDC-Fed mice. k ELISA of TGFβ1 and Mmp14 in coculture supernatants from direct and indirect MDO–JS-1 coculture systems. l ELISA analysis of TGFβ1, α-SMA, and Col1a1 secretion in coculture supernatants. m, n ELISA of HGF secretion in mouse serum (n = 3) and HCC samples (n = 5). The data are shown as the means ± SEMs (n = 3 or 5). *p < 0.05; **p < 0.01; ***p < 0.001

模块 5：HGF/MET 通过 PI3K/AKT-NFκB、STAT3 通路上调 UbD 驱动 MDB 形成

1.研究逻辑：HGF 刺激、MET/PI3K 抑制剂阻断正反实验，蛋白磷酸化验证下游通路；3D 类器官免疫荧光验证通路活性；明确 NF-κB、STAT3 结合 UbD 启动子促进转录。

2.核心实验：Hepa1-6 细胞 TNFα/IFNγ 造模；梯度 HGF 给药、抑制剂干预；MET/AKT/IκB/STAT3 磷酸化 WB；MDB 类器官 MET 抑制剂处理免疫荧光；UbD 启动子转录结合位点生信预测。

3.关键实验结果：

• HGF 刺激激活 MET 磷酸化，依次激活 AKT、IκB、STAT3，上调 Ub、TNFα；MET 或 PI3K 抑制剂可完全逆转该效应；
• 3D 类器官阻断 MET 后，p-AKT、p-IκB、STAT3、UbD 表达下降，类器官萎缩、MDB 标志物减少；
• UbD 启动子存在 NF-κB p50、STAT3 特异性结合位点，炎症因子协同放大 UbD 转录。

Fig. 6 The HGF/MET axis drives MDB formation via NF-κB and STAT3 signaling. a Experimental design schematic. Hepa1-6 cells were stimulated with TNFα (40 ng/mL) and IFNγ (400 ng/mL) at three-day intervals, and the cells were collected for assays. b mRNA expression in Hepa1-6 cells at 24, 48, and 72 h after HGF (20 ng/mL) treatment. c Western blot analysis of total and phosphorylated MET, AKT, and IκBα in MDB-forming Hepa1-6 cells after HGF treatment. d, e Western blot analysis of PI3K/AKT/NF-κB signaling in Hepa1-6 cells treated with HGF in the presence/absence of a PI3K inhibitor (LY294002) or MET inhibitor (PF-02341066). f Western blot analysis of signaling proteins in MDB-HCC tissues. g Immunofluorescence costaining of p-AKT, p-MET, and p-IκBα with p62 in MDB-HCC versus nontumor tissues (n = 5). Scale bar: 50 μm. h Bright-field and immunofluorescence imaging of MDOs treated with the MET inhibitor revealed that K8/p-AKT/p-IκBα colocalized with p62. Scale bars: 200 μm (bright field) and 50 μm (fluorescence). i Immunofluorescence imaging of STAT3 and UbD colocalization in MDOs treated with MET inhibitors. Scale bars: 100 μm (fluorescence). The data are shown as the means ± SEMs (n = 3 or 5). *p < 0.05; **p < 0.01; ***p < 0.001

模块 6：UbD 敲除（UbD?/?）小鼠体内反向验证通路功能

1.研究逻辑：CRISPR 构建 UbD 全身敲除小鼠，DDC 单 / 联合 CCl?诱导纤维化，对比野生型明确 UbD 缺失对 HGF/MET、炎症、纤维化、MDB 的抑制作用。

2.核心实验：UbD?/?小鼠鉴定；WB/qPCR 检测通路、MDB、纤维化基因；HGF/TNFα ELISA；组织免疫荧光 Vimentin/Desmin；多组病理染色量化纤维化、脂变、MDB。

3.关键实验结果：

• UbD 敲除显著抑制 MET 表达、降低 HGF 与 TNFα 分泌；纤维化标志物 α-SMA、胶原、Vimentin 大幅下调；
• UbD?/?小鼠肝脏 MDB 包涵体、脂肪沉积、胶原纤维显著减少；CCl?联合损伤下保护效应更突出；
• HSC 活化、EMT 转化被显著抑制，完整证明 UbD 是 HGF/MET 通路下游执行分子。

Fig. 7 UbD deletion suppresses MDB formation and fibrosis. a Schematic of the generation of UbD knockout (UbD?/?) mice via CRISPR/Cas9. b Western blot (top) and RT?qPCR (bottom) analyses of UbD, p62, K8, and proteasome-related genes in WT-DDC vs UbD?/?-DDC mice. c Western blot analysis of the HGF/MET/NF-κB and STAT3 pathways in WT vs UbD?/? mice. d ELISA analysis of HGF, TNFα, IFNγ, and IL-6 secretion in WT and UbD?/? mice after 8 weeks of DDC treatment (n = 3). e, f RT?qPCR of fibrosis markers (Mmp14, Mmp2, Acta2, Col1a1, Col3a1, and Col4a1) and EMT markers (Cdh1, Cdh2, and Vim) in WT and UbD?/? mice. g The expression of MDB and the fibrosis-related molecule vimentin was detected by Western blot in the UbD- / - -/DDC-CCl4 mice. h Immunofluorescence showing colocalization of Vimentin and UbD (white arrows) in the UbD?/? and WT mice. Scale bar: 50 μm; zoom: 10 μm. i H&E, Masson, Oil Red O and α-SMA immunostaining showing reduced MDB formation (yellow arrows), fibrosis, and lipid accumulation in the UbD?/?-DDC mice. Scale bar: 100 μm. j Histological analysis of liver sections from DDC-CCl?-induced fibrosis models (WT vs UbD?/?). Scale bar: 100 μm. The data are shown as the means ± SEMs (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 8 Schematic diagram of the study. Functional in vitro and in vivo experiments demonstrated that, upon injury, HGF interacts with c-Met, activating the PI3K/AKT/NF-κB and STAT3 pathways and promoting UbD upregulation and the release of the proinflammatory cytokine TNFα, which leads to MDB pathogenesis and fibrosis development

五、Absin（abs9516 小鼠肝脏类器官培养试剂盒）产品整体作用总结

1. 搭建不可替代 3D 病理体外模型：本研究首次建立 DDC 诱导 MDB 类器官（MDO）体系完全依托 Absin abs9516 试剂盒，传统二维细胞无法模拟肝细胞与 HSC 空间旁分泌交互，试剂盒配套培养基、消化缓冲、基质胶配套方案可稳定复刻体内 MDB 蛋白聚集、纤维化因子分泌病理特征；
2. 支撑药物干预与通路功能体外验证：试剂盒培养的 MDB 类器官可稳定传代、冻存复苏，重复性强，用于 MET 抑制剂梯度干预实验，直观可视化 HGF/MET 通路阻断后 MDB 形成抑制现象，为体内动物实验提供前置体外药效证据；
3. 打通细胞 - 类器官 - 动物多层次实验链条：衔接体外肿瘤细胞、3D 类器官、DDC/UbD 基因小鼠三层体系，完整串联细胞分子机制与体内病理表型，满足 STTT 高分期刊对多层次模型验证的严苛要求，为 HGF/MET/UbD 作为肝病靶点结论提供关键体外类器官数据支撑。