骨髓间充质干细胞NRF2低表达在移植后血小板延迟植入中的作用及机制研究

Prolonged isolated thrombocytopenia (PT) is a common and serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathogenesis of PT remains unclear and there were no effective prevention and treatment. In recent years, the applicant has been making her continuous efforts on the pathogenesis and the novel treatment strategies for PT after allo-HSCT. Our previous studies demonstrated that the number and function of megakaryocytes were abnormal in PT patients, and the decrease and dysfunctional of bone marrow mesenchymal stem cells (MSCs) hampered the number and function of megakaryocytes, which ultimately lead to the occurrence of PT after allo-HSCT. Meanwhile, the damage of bone marrow MSCs may be related to the high levels of intracellular reactive oxygen species (ROS) level in PT patients. Nuclear factor E2-related factor 2 (NRF2) is a key molecule regulating the level of ROS in cells. So far, whether the abnormalities of NRF2 in bone marrow MSCs affect the number and function of megakaryocytes, and the role of NRF2 abnormality in the pathogenesis of PT and its mechanism have not been reported yet. More recently, our preliminary data showed the decreased expression of NRF2 in bone marrow MSCs of PT patients comparing with that of patients with good graft function. Therefore, based on our previous work and preliminary experiments, we will clarify the expression characteristics of NRF2 in bone marrow MSCs of PT patients and explore whether the low expression of NRF2 in bone marrow MSCs affects the number and function of megakaryocytes through ROS, which ultimately lead to the occurrence of PT.

血小板延迟植入（PT）是异基因造血干细胞移植后常见而严重的并发症，其发病机制尚未完全阐明，无有效防治手段，是亟待解决的重要临床科学问题。近年来，申请人围绕PT发病机制开展了系列研究，发现：①PT患者骨髓巨核细胞数量减少和功能异常。②骨髓间充质干细胞（MSCs）损伤影响巨核细胞数量和功能，参与了PT发生。③活性氧增多是PT患者骨髓MSCs损伤的重要机制。核因子E2相关因子2（NRF2）是调控细胞活性氧水平的关键分子。迄今为止，骨髓MSCs内NRF2异常是否影响巨核细胞数量和功能，及NRF2异常在PT发病机制中的作用及其机制尚未见报道。申请人近期预实验发现：PT患者骨髓MSCs内NRF2表达低于植入良好患者。因此，在前期工作和预实验基础上，本课题将明确PT患者骨髓MSCs内NRF2表达特征，探讨骨髓MSCs内NRF2低表达是否通过活性氧影响巨核细胞数量和功能，从而导致PT发生。

研究背景：.活性氧增多是移植后血小板延迟植入(PT)患者骨髓MSCs损伤的重要机制。核因子E2相关因子2（NRF2）是调控细胞活性氧水平的关键分子。目前骨髓MSCs内NRF2异常是否影响巨核细胞数量和功能及NRF2异常在PT发病机制中的作用及其机制尚未见报道。.主要研究内容：.①通过前瞻性病例配对研究和骨髓间充质干细胞体外实验，明确血小板延迟植入患者骨髓间充质干细胞内NRF2及其下游调控的抗氧化蛋白和酶是否存在差异。.②通过体外实验降低植入功能良好患者骨髓间充质干细胞内NRF2表达， 并对NRF2低表达的骨髓间充质干细胞进行修复研究，探讨NRF2变化对ROS水平的调控作用，对骨髓间充质干细胞和巨核细胞的影响。.③ 应用NRF2激动剂对血小板延迟植入患者骨髓间充质干细胞进行体外修 复研究，观察骨髓间充质干细胞内ROS水平、骨髓间充质干细胞及巨核细胞数量和功能的变化情况。.重要结果：.①证实了异基因造血干细胞移植后血小板延迟植入组间充质干细胞内NRF2及其下游调控的抗氧化蛋白表达低于植入功能良好患者。.②在体外用NRF2激动剂TBHQ 通过降低血小板延迟植入患者骨髓间充质干细胞内ROS水平、改善骨髓间充质干细胞，从而改善巨核细胞功能。.关键数据及其科学意义：.本研究揭示了骨髓间充质干细胞NRF2调控可能为血小板延迟植入的发病机制之一。首次尝试在体外用NRF2激动剂TBHQ 通过降低血小板延迟植入患者骨髓间充质干细胞内ROS水平、改善骨髓间充质干细胞，从而改善巨核细胞功能，为将来移植后血小板延迟植入患者制定临床防治新策略。本课题初步阐明或部分阐明血小板延迟植入这一移植后严重并发症的发病机制，尝试找到了血小板延迟植入可能的新的治疗方案，为将来提高血小板延迟植入患者的临床诊疗水平，对于改善异基因造血干细胞移植患者的临床预后具有重要意义。