低能激光对造血干细胞的电离辐射防护作用及机理研究

Ironizing radiation could induce radiation injury such like increasing reactive oxygen species (ROS), DNA damage, and apoptosis of mitochondria pathway, and brought serious sickness and disaster in radiation accidents. Therefore, it will be meaningful for exploring new methods without toxic and side effects in radiation protection study. Low level laser irradiation (LLLI) was a kind of light that was harmless to biological tissue, and performed function of irradiation protection by photo-modulation. Mechanism of the LLLI in anti-irradiation was not clear. Its function might be initially regulated by ROS increasing with a low level dose, and promoted phosphorylated regulation in activation of PKC regulation, hydrolyzed in keap1-Nrf2 compound protein， released Nrf2 access into nuclear and banded onto antioxidant6 responsive element (ARE), initiated the transcription of phase Ⅱ antioxidant enzyme genes，leading an enhanced activity of irradiation protection. Therefore, We would use radio-sensitive tissue hematopoietic stem cells as research object, and employ ROS cleaning regent NAC and Nrf2 inhibitor Brusatol (functioned by degradation of Nrf2 protein) in in vitro experiments (Eliasa, Westernblot and SAGE et al.), using Nrf2 knockdown animal in assessment of anti-radiation level in vehicle control and Nrf2 knockdown mice after intravascular blood irradiation (cell counting of blood, nucleated cell counting in marrow, bone marrow smear, pathological section and survival time.), besides, we also analyzed treatment effects in transplantation of stem cells preprocessed using LLLI into mice with hematopoietic function acute radiation sickness (H-ARS). Exploring the key pathway in anti-radiation function of LLLI through in vitro and in vivo experiments. Proposed the possible pathway ROS-Nrf2-ARE in anti-radiation function of LLLLI. Accomplishment of this study might verified the irradiation antioxidant protection of LLLI, provided regulation pathway in theoretical support aspect. It would be not only using for radiation protection and stem cell transplantation, but also in oxidant related disease such like Parkinson.

探索新的无毒副作用的辐射防护方法具有重要意义，低能激光照射（LLLI）是一种对生物机体无毒无害的光照，其引起的生物光调节作用对辐射诱导的损伤有一定防护作用，但作用机理尚未明确，我们根据前期研究和相关报道推测，可能作用机制为LLLI引起细胞低水平ROS应激，经由Nrf2-ARE途径产生的抗氧化酶活性增加而产生抗氧化作用的辐射防护效应。因此，本研究利用辐射敏感组织造血干细胞，拟采用ROS清除、Nrf2抑制、Nrf2敲除动物、血管内激光血液照射后的抗辐射评价、激光照射处理移植干细胞的急性放射造血功能损伤小鼠的疗效分析等方法，从体内外实验中探索LLLI诱导的辐射防护作用是经ROS-keap1-Nrf2-ARE途径发挥作用。本项目完成可在理论上阐明LLLI的抗氧化辐射防护作用机制，为LLLI在辐射防护、移植干细胞的功能增强以及其他氧化损伤相关的神经退行性疾病等治疗中的应用，提供新的理论和治疗思路。

本研究通过LLLI对干细胞的生物学作用研究, Nrf2通路与LLLI介导的干细胞抗辐射作用研究以及LLLI预处理干细胞在辐射损伤救治中的体内应用来讨论LLLI在辐射损伤防护中的作用, 应用前景及分子机理。主要讨论了细胞存活率、DNA损伤评价、ROS水平、电离辐射防护作用等辐射防护相关的指标。干细胞的低剂量激光氧化应激的作用能够提高抗辐射防护作用。LLLI预处理的细胞抗氧化酶活性均有增加，降低了电离辐射的敏感性。在研究其发挥作用分子作用机理中，选取了C57小鼠进行了辐射造血损伤模型，LLLI引起Nrf2的高表达。因此，采用ROS清除、Nrf2干扰、激光照射处理移植干细胞的急性放射造血功能损伤小鼠的疗效分析等方法，对LLLI发挥作用的可能通路ROS-keap1-Nrf2-ARE进行验证。蛋白印记方法结果显示Nrf2的蛋白高表达，其对应LLLI干扰后一些抗氧化酶水平升高，Nrf2干扰实验也影响了LLLI发挥抗辐射作用的能力。另一方面，体外干细胞移植后，从血细胞计数、骨髓有核细胞计数、骨髓涂片、病理切片以及生存周期上进行分析。LLLI预处理的MSC移植小鼠骨髓脾脏病理组织变化明显，造血功能、增生程度积分明显增高。造血组织容量改善更好。Nrf2干扰的干细胞经LLLI预处理与未激光预处理组并无显著差异。理论上阐明LLLI的抗氧化辐射防护作用机制，为LLLI在辐射防护、移植干细胞的功能增强以及其他氧化损伤相关的神经退行性疾病等治疗中的应用，提供新的理论和治疗思路。