褪黑素抗早衰效应在干细胞修复骨缺损中的应用及SIRT1介导机制的研究

Treating large bone defects remains a major challenge in orthopaedic surgery. Bone tissue engineering strategies with stem cell now offer a promising solution to the drawbacks of traditional bone grafting techniques. Inflammatory and hypoxic microenvironment in defect site causes oxidative stress-induced premature senescence of transplanted mesenchymal stem cells (MSCs), irreversibly inhibits their proliferative ability, and suppresses their regenerative potentials. We previously reported that melatonin could protect MSCs from premature senescence induced by H2O2. This result was made evident by improved cell proliferation, decreased senescence-associated β-galactosidase activity and the expression of senescence-associated genes. Our recent study revealed that the protective effects of melatonin might be closely related to SIRT1-dependent pathway. However, the underlying mechanisms are unknown. Hence, we propose that melatonin could protect MSCs from premature senescence. In this project, MSCs will be treated in vitro with H2O2 to induce oxidative stress-induced premature senescence, and the protective effects of melatonin on reversing MSCs senescent phenotypes in a dose-dependent manner will be studied. We will focus on the critical role of SIRT1-dependent pathway in protective effects of melatonin. Also, we will analyze whether in vitro and in vivo osteogenic potential of MSCs could be recovered by treating with melatonin, with the purpose for enhancing bone tissue regeneration. Results will offer important information for theory of preventing MSCs senescence, direct the design of anti-senescent biomaterials, and improve the clinical therapy effects of MSCs in bone defects repairing.

骨缺损修复一直是骨科亟待解决的难题，以干细胞为基础的骨组织工程将改变传统“以创伤修复创伤”的骨缺损治疗模式。但缺损部位的炎症、缺氧微环境易导致干细胞发生早衰，造成不可逆的增殖停滞并破坏其组织修复功能。课题组前期工作发现褪黑素能有效抑制干细胞早衰，表现为衰老染色阳性细胞比率减少并且p16等衰老相关基因表达降低，进一步研究表明褪黑素抗早衰效应与SIRT1密切相关，但褪黑素如何调控SIRT1功能并由此激活细胞内的抗早衰机制尚未明确。因此我们提出“褪黑素通过SIRT1预防干细胞早衰”这一假设。拟诱导干细胞早衰，分别从维持干细胞增殖能力和保护成骨分化功能两个角度，明确褪黑素抗早衰功能及浓度依赖性，阐明SIRT1在抗早衰机制中的关键介导作用，动物实验验证褪黑素对干细胞骨修复的抗早衰保护作用。研究成果将有助于明确干细胞早衰的防治理论，指导设计具有抗衰老性能的生物材料，提高干细胞技术治疗骨缺损的实际效果。

项目背景：骨缺损修复一直是骨科亟待解决的难题，以干细胞为基础的骨组织工程将改变传统“以创伤修复创伤”的骨缺损治疗模式。但缺损部位的炎症、缺氧微环境易导致干细胞发生氧化应激性早衰，造成不可逆的增殖停滞并破坏其组织修复功能。因此当面临氧化应激时，如何预防MSCs早衰、妥善修复DNA损伤和保持其组织再生能力是目前骨组织工程中亟待解决的关键问题。..主要研究内容与重要结果：首先，我们利用200μM H2O2诱导BM-MSCs早衰但不会造成细胞凋亡，建立早衰模型。加入不同浓度褪黑素干预，研究不同浓度的褪黑素对早衰MSCs增殖和成骨分化能力的调控作用。结果发现褪黑素干预能抗BM-MSCs早衰，提高早衰BM-MSCs增殖活性，减少SA-β-Gal衰老染色阳性率，处于S期的细胞比例明显上升，提高基质矿化程度和成骨分化相关基因表达，并且这种作用趋势具有浓度依赖性。其次，我们重点考察了褪黑素受体介导的SIRT1信号分子在褪黑素抗早衰中的信号传导机制。褪黑素降低了衰老调控因子p16INK4α的基因和蛋白表达水平、p38MAPKs的磷酸化水平，并提高SIRT1的基因和蛋白表达水平。但是加入Sirtinol或Luzindole均减弱了褪黑素的抗早衰作用。褪黑素受体介导的SIRT1信号通路在褪黑素抗早衰中发挥关键介导作用。最后，建立大鼠颅骨缺损模型，诱导早衰MSCs成骨分化，与骨支架修复材料复合后植入缺损部位，在此期间对大鼠进行褪黑素注射干预。经褪黑素干预的MSCs/支架复合物在体内具有良好的成骨性能，褪黑素能有效提高MSCs修复骨缺损的实际效果。 ..关键数据及其科学意义：研究了褪黑素具有保护早衰MSCs增殖能力和成骨分化功能的作用；揭示了SIRT1信号分子在褪黑素抗早衰机制中的关键介导作用；阐明了褪黑素提高早衰MSCs体内修复骨缺损的实际效果。..项目执行期间，共计发表SCI收录论文14篇（均带项目号），申请发明专利2项，其中1项获得授权，参加国内重要学术会议3人次。