脐带间充质干细胞来源外泌体调控靶细胞应答抑制YAP活性延缓糖尿病肾病的作用及机制研究

Our previous studies have proved that human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) have shown promising results in renal, skin, liver and myocardial injury model, can be exploited as powerful tool in regenerative medicine. The prevention and therpty of diabetic kidney diseases(DKD) is an important part of the health China 2030 program. We have found that hucMSC-Ex can significantly decrease the level of blood glucose and delay the occurrence in DKD models.The function of hucMSC-Ex may be related to promote YAP cytoplasmic retention and ubiquitination degradation. However, the role and mechanism of hucMSC-Ex in DKD remains unclear. Therefore, this study will use CRISPR/cas9 gene editing, single cell sequencing, LC-MS/MS, co-immunoprecipitation and other cellular and molecular biology techniques, combined with YAP specific phosphorylation site gene mutation mouse model, investigate the mechanism of hucMSC-Ex on the prevention and treatment of diabetic kidney diseases. We will identify the key molecules and associated signaling pathways for regulating YAP phosphorylation by hucMSC-Ex, elucidate the response pathway of the renal tissue target cells and provide a new strategy to prevent and treat DKD.

课题组前期研究表明脐带间充质干细胞来源外泌体（hucMSC-Ex）对肾、皮肤、肝、心肌等组织损伤模型有良好的修复效果，显示在再生医学领域的广阔应用前景。糖尿病肾病（DKD）的防控是健康中国2030规划的重要内容，预实验结果发现hucMSC-Ex能够显著地降低糖尿病肾病（DKD）模型鼠的血糖并延缓其病程，可能是通过磷酸化依赖途径促进了YAP蛋白的胞质内滞留和泛素化降解，进而抑制其生物学活性，但确切的分子机制亟待阐明。因此，本研究将采用CRISPR/cas9基因编辑、单细胞测序、LC-MS/MS、免疫共沉淀等技术，结合YAP特定磷酸化位点突变小鼠模型，深入探讨hucMSC-Ex延缓DKD的作用及机制，明确hucMSC-Ex调控YAP磷酸化的关键分子及相关信号通路，揭示hucMSC-Ex作用的肾组织靶细胞及靶细胞对外泌体的应答，为糖尿病肾病的预防和治疗提供新策略。

项目研究背景：.糖尿病肾病（DKD）的防控是健康中国2030规划的重要内容，而脐带间充质干细胞来源外泌体（hucMSC-Ex）在组织损伤模型有良好的修复效果，探究hucMSC-Ex对慢性肾病DKD的修复和治疗机制。本项目主要探讨hucMSC-Ex抑制靶细胞YAP信号通路延缓糖尿病肾病进程的作用及机制，为揭示hucMSC-Ex抑制肾纤维化治疗提供新的分子靶点和理论依据。.项目主要研究内容：.研究脐带间充质干细胞来源外泌体（hucMSC-Ex）对糖尿病肾病的干预作用；确定HucMSC-Ex通过抑制YAP信号通路延缓糖尿病肾病进程的作用；筛选及确定HucMSC-Ex抑制YAP的关键分子；基于转录组测序阐明hucMSC-Ex作用糖尿病肾病的靶细胞及其应答机制。.项目关键数据：.糖尿病肾病中持续高糖血症通过活化TGF-β1/Smad2/3/YAP信号轴促进系膜细胞纤维化样改变，高糖刺激系膜细胞Smad2/3和YAP入核增加，肾组织病理HE和Masson染色显示肾小球基底膜厚度明显增加，α-SMA 表达增加，大量胶原纤维沉积加速DKD肾间质纤维化。而hucMSC-Ex能定向迁移至受损的肾组织，显著抑制胶原沉积改善肾功能延缓DKD疾病进程。体外实验hucMSC-Ex处理抑制系膜细胞HBZY-1中YAP水平降低α-SMA 表达。免疫荧光证实hucMSC-Ex处理后胞质内Ser381-YAP、Ser127-YAP明显上调，总YAP水平降低，显示hucMSC-Ex可能通过磷酸化依赖途径促进YAP蛋白胞质内滞留和泛素化降解。免疫共沉淀实验显示YAP结合的泛素分子明显增多发生泛素化降解。质谱数据发现hucMSC-Ex内富含CK1δ/β-TRCP激酶泛素系统，促进YAP泛素化降解。腺病毒敲减间充质干细胞CK1δ/β-TRCP，其hucMSC-Ex抑制DKD肾间质纤维化作用减弱。证实hucMSC-Ex递送CK1δ/β-TRCP激酶泛素系统调控YAP泛素化降解。进一步基于转录组测序分析发现hucMSC-Ex作用的靶细胞及调控靶细胞应答，抑制纤维化样改变延缓糖尿病肾病纤维化进程。.项目科学意义：.本研究不仅阐明了hucMSC-Ex抑制YAP延缓糖尿病肾病的关键活性分子，而且明确了其靶细胞应答通路为防治DKD 提供新思路，具有重要的理论意义和潜在应用价值。