Notch1信号通路在炎症微环境诱导间充质干细胞老化中的作用及机制研究

Mesenchymal stem cells (MSC) serve as important tools for cell therapy in clinic, owned to their functions in immunoregulation, mulitple lineage differentiation potential as well as support of hematopoiesis. However, senescence can impair or inhibit therapeutic function of MSC. Therefore, it is of great importance to investigate senescence of MSC and its potential mechanisms. Our previous study showed that infusion of in vitro expanded MSC couldn’t ameliorate endotoxemic tissue injuries, and they could even aggravate the inflammatory response. We asked whether in vivo inflammatory microenvironment could impair biological functions of MSC through induction of cell senescence, and what is the potential mechanism. These questions remains to be clarified till now. For this reason, we try to mimic the in vivo inflammatory microenvironment with serum from endotoxemia models, and investigate effects of inflammatory microenvironment on senescence of MSC. Then, we will focuse on Notch1 signaling pathway to clarify the role of Notch1 signaling in inflammation-induced MSC senescence, by making use of RNA silencing and overexpression technique. Finally, we will investigate whether sencescence-associated SIRT1 and transcription factor NF-κB、C/EBPβ can regulate MSC senescence in the upstream and downstream of Notch1 respectively. The main purpose of this study is to clarify the effects of inflammatory microenvironment on MSC senescence and the mechanisms underlined. It will provide experimental and theoretical evidence for exploring ideal means to optimize quality of MSC, and thus to obtain better outcomes in clinical trials of cell therapy.

间充质干细胞（MSC）具有免疫调节、多向分化及造血支持等功能，是临床细胞治疗的重要工具，但细胞老化能削弱甚至抑制MSC的治疗作用。因此，研究MSC老化及机制具有重要临床价值。我们前期研究发现，静脉输注体外扩增的MSC不能缓解内毒素血症模型的组织炎性损伤，甚至有加重趋势。那么，体内炎症微环境是否通过诱导MSC老化而损伤MSC的正常功能？其潜在机制是什么？至今尚无相关的系统研究报道。为此，我们将利用内毒素血症模型血清模拟体内炎症微环境，探讨其对MSC老化的影响；并以Notch1信号通路为切入点，结合基因过表达和沉默技术，研究Notch1通路活化在炎症微环境诱导MSC老化中的作用；进一步研究老化相关分子SIRT1和转录因子NF-κB、C/EBPβ是否分别在Notch1通路的上、下游共同调节MSC老化。本研究旨在阐明炎症微环境对MSC老化的作用及机制，为寻找优化MSC质量的方法提供实验和理论依据。

间充质干细胞（MSCs）具有免疫调节功能，可被用于治疗炎性及免疫相关疾病。但MSCs在免疫治疗中的效果有赖于其活性及功能状态的正常。细胞衰老是降低细胞活性和功能、降低细胞治疗效果的重要因素，而炎症是诱导细胞衰老的原因之一。那么，体内炎症微环境是否影响MSCs的生物学功能或诱导其衰老？为此，本研究探讨了脓毒症炎症微环境对骨髓MSCs(BMSCs)生物学功能和衰老的影响，并探讨相关分子机制。研究首先探讨了脓毒症微环境对体内组织中MSCs的影响。结果发现，与对照组比较，脓毒症模型来源的BMSCs增殖能力显著降低，迁移能力增强，细胞内衰老相关分子如β-gal的活性增加、衰老相关分泌因子的表达增强、细胞周期阻滞。继而，研究利用脓毒症动物血清刺激正常BMSCs，以模拟细胞治疗中体内炎症微环境对输注入体内的MSCs的刺激。结果发现，炎性血清刺激后，BMSCs增殖能力显著下降并且细胞具有衰老表现。研究进一步探讨了Notch通路在该过程中的作用，结果发现，炎性微环境诱导Notch1通路活化，并且外源性阻断Notch通路后，细胞衰老相关表现被部分逆转。同时，炎症微环境能抑制SIRT1的表达，并且促进Notch1与SIRT1的共定位，但Notch1的乙酰化水平增高，提示SIRT1对Notch1的去乙酰化作用下降。另一方面，炎性血清诱导Notch1下游C/EBPβ的活化，但对NF-κB的活化无显著影响；阻断Notch通路后，炎性血清诱导的C/EBPβ的活化作用亦减弱。这些结果提示，脓毒症炎性微环境可改变BMSCs的生物学功能并诱导其衰老，该作用与SIRT1-Notch1-C/EBPβ通路有关。.本研究结果能为临床研究中直接利用MSCs产品用于脓毒症/内毒素血症等炎性病变的治疗存在的风险提供理论和实验参考，并为无细胞性MSCs衍生物（分泌组）如外泌体在炎性病变中的应用优势提供了科学依据。