海带多糖通过调节vWF表达，保护放射性脑损伤的研究

Radiation brain injury is a common and serious clinical complication after radiation therapy for brain and neck tumor, and seriously troubles clinicians for a long time. Our previous study indicated that secondary brain injury was mainly caused by injured endothelial cells after radiation, but its mechanism remain unclear. In addition, our research further revealed that laminaria polysaccharide significantly improved memory disorders of mouse following radiation. In rats model with endothelial cells injuried by adrenaline, laminaria polysaccharide was known to reduce the expression of von Willebrand factor (vWF) secreted by endothelial cells, and played a role in ant thrombosis. Therefore, we hypothesize that laminaria polysaccharide maybe regulate the expression of vWF , and offer neuroprotection for radiation-induced brain injury. To confirm this view, rat's model with radiation-induced brain injury will be established in vitro and in vivo. Following laminaria polysaccharide, neurological behavior, cell and molecular biology technique, and so on, will be employed to observe the expression of vWF, thrombosis , and the changes of nerve cell and cerebral function. we will come up with the molecular mechanism of laminaria japonica polysaccharides in protection of radiation brain injury by analyzing the relationship among vWF , thrombosis and nerve cell . After this research, a theoretical basis of laminaria polysaccharide in prevention and treatment of radiation brain injury will be provided for physicians.

放射性脑损伤是头颈部肿瘤放射治疗过程中最常见的并发症，长期以来严重地困扰着临床医生。我们的前期工作表明，放射致内皮细胞损伤是脑继发损伤的主要原因，但其机制尚未清楚。我们进一步发现海带多糖可显著改善放射性脑损伤的记忆功能障碍。在肾上腺素致内皮损伤大鼠模型中，海带多糖可通过下调内皮细胞分泌的血管性血友病因子（vWF）拮抗内皮损伤大鼠血栓形成。因此，海带多糖也很可能通过影响vWF的表达对放射性脑损伤起保护。为证实这一观点，本项目拟建立放射性脑损伤的大鼠模型，采用神经行为学、细胞和分子生物学等技术手段，从整体和细胞水平，观察vWF表达、血栓形成、神经细胞变化和干预效果，分析vWF与血栓形成和神经细胞病理变化的关系, 探讨海带多糖对放射性脑损伤保护的分子机制，为海带多糖运用于临床防治放射性脑损伤提供理论依据。

放射性脑损伤的修复和再生一直困扰临床医生，据此，本研究从成体层面建立放射诱导脑损伤模型，采用Morris水迷宫观察海带多糖干预前后小鼠记忆改变。神经行为结束后，检测血浆中FIB、APPT、PT、TT等凝血因子含量和vWF表达，分析放射因素和海带多糖干预在凝血中作用。采用ELESA检测脑内IL-1β、TNF-α等炎性因子含量；采用免疫组化手段观察脑内Collagen Ⅳ、Iba-1、GFAP和NSE等表达，齿状回和室管膜下区神经发生；电镜下观察内皮细胞、神经胶质细胞、神经元和髓鞘变化以及脑内微血栓的形成，分析放射因素和海带多糖干预对神经细胞和血栓形成的影响，结果发现放射不仅能损伤内皮细胞和神经元，抑制神经发生，同时促进脑内血栓形成和炎性反应，而海带多糖能改善放射诱导损伤的脑功能，保护神经元和神经干细胞，同时也保护脑内血管内皮细胞，抑制血栓形成和炎性发生，然而其机制尚明确。为了进一步明确其机制，本项目分离和培养大鼠脑血管内皮细胞、神经胶质细胞和神经元，建立放射诱导损伤的细胞模型，光镜下观察内皮细胞vWF+表达、神经元NSE+表达和胶质细胞GFAP+表达，采用ELASA分别检测内皮细胞培养液vWF、t-PA和PAI-1因子含量，神经元培养液中Bax和BCL-2因子含量，以及神经胶质培养液中Bax、BCL-2、TNF-a因子含量，分析放射因素和海带多糖干预对血管内皮细胞、神经胶质细胞和神经元作用及其机制。结果发现：①放射是内皮细胞损伤的因素，其能促进血管内皮细胞释放vWF、PAI-1 因子，抑制t-PA 的释放，而LJP具有抑制vWF、PAI-1 因子释放，促进t-PA 因子释放，保护内皮细胞，抑制血栓的形成；②海带多糖具有保护神经元作用，其机制可能与LJP 通过促进神经元Bcl-2 表达和抑制Bax表达有关。