T2A中GalT I和V对创伤性脑损伤的再生修复机制

Central nervous system injury is a cause of major neurological disability, and no satisfactory treatment is currently available. This item will combine the cell transplantation and gene therapy, type 2 astrocytes (T2A) intervened the expression and activity of β-1,4-galactosyltransferase I and V (GalT I and V) could suppress the activation of T1A, thereby inhibit glial scar formation, which was regarded as a breakthrough. The aim is to study the regeneration and repair mechanisms of GalT I and V in T2A in central nervous system injury. First of all, in vitro, to study the effect of active T1A or GalT I/V intervention in T2A in the gene level on biological behavior including migration and adhesion, and to reveal the transcription regulation mechanisms of GalT I and V in T2A.Secondly, Followd by intervention of GalT I and V expression and activity in T2A, to observe its effects on T1A activation, neuronal injury, neurite growth.Further, in vivo, to investigate the biological function of GalT I and V in T2A in vivo, and tranplanting T2A intervened GalT I and V expression into the brain damage area, to observe the role which will play in the central nervous system regeneration and repair process.Finally, in the overall level, establishing the transgene rats which GalT I and V expression will be intervened in T2A targetly,then making brain injury model, to study the function of T2A and GalT I and V in the central nervous system regeneration. Completing the present study will provide a new way for treating the central nervous system injury and provide the reliable experimental and theroretical basis.

中枢神经系统损伤目前尚无令人满意的治疗方法，本项目将细胞移植和基因治疗结合起来，以干预GalT I/V表达的2型星形胶质细胞（T2A）可抑制T1A活化，从而抑制胶质瘢痕的形成入手，研究T2A中GalT I/V在中枢神经系统损伤后的再生修复机制。首先在细胞水平，研究T1A活化或在基因水平上干预T2A中GalT I/V的表达对T2A迁移、黏附等生物学行为的影响，阐明GalT I/V在T2A中的转录调控机制；其次观察干预GalT I/V的表达及活性的T2A对T1A的活化、神经元损伤与突起生长的影响；最后在整体水平，观察GalT I/V在体内T2A中的生物学作用，并将干预GalT I/V表达的T2A移植入脑损伤区域，或构建靶向干预T2A中GalT I/V的转基因鼠，研究T2A中GalT I/V在中枢神经再生过程中的作用。课题的完成将为开辟中枢神经损伤新的治疗途径提供可靠的实验基础和理论依据。

星型胶质细胞是中枢神经系统中最主要的细胞，创伤性脑损伤(traumatic brain injury, TBI)目前尚无令人满意的治疗效果。本研究在细胞水平通过A2B5和GFAP的共定位分离鉴定1型和2型星形胶质细胞，在分子水平，发现1型星胶炎性因子作用下可通过自分泌方式产生的TNFα影响GalT I和GalT V的表达。2型星胶中TNFα通过TNFR1和TNFR2由细胞核向细胞浆和细胞突起的转位诱导GalT I的表达，且TNFα可通过自分泌循环通路在炎症反应过程中发挥作用。星胶划伤后GalT I的表达呈时间依赖性上调，GalT I抗体封闭可抑制星胶的迁移，PQQ可恢复损伤星胶的增殖和迁移能力，PQQ可通过抑制GalT I的表达来影响PC12细胞、SHSY5Y细胞的细胞周期。构建GalT I和V siRNA，以及GalT V干扰和过表达慢病毒载体，筛选稳转细胞株，观察GalT I和V对细胞生物学行为的影响。过表达GalT V可促进C6细胞的增殖和迁移能力，而干扰GalT V则能抑制C6细胞的增殖和迁移能力。PQQ对于Glutamate造成的星形胶质细胞损伤及过氧化物对PC12细胞的损伤具有一定的保护功能，具有促进增殖和迁移的作用，其机制可能是通过抑制凋亡和自噬发挥作用的。整体水平，TBI后PQQ通过对GalT I和V的影响发挥神经保护作用。大鼠TBI后PQQ可明显地改善学习记忆及空间辨别能力，恢复脑电生理功能，浓度依赖性上调皮层区和海马CA2区的GalT I, V和Gal β-1,4-GlcNAc group的表达水平。TBI后在神经元和星形胶质细胞中GalT I在自噬和凋亡的调节过程中发挥重要作用，WISP1参与TBI后细胞的自噬过程，PQQ通过 PI3K/AKT信号通路对WISP1进行调节，但不直接参与cleaved-Caspase3的细胞凋亡途径，WISP1 siRNA对星形胶质细胞具有促进增殖、抑制迁移的作用。将干预了GalT V表达的病毒载体通过微量注射泵缓慢注入脑损伤区域，发现干扰了GalT V的病毒载体抑制迁移，过表达GalT V则促进迁移。通过旋转运动测试和Morris水迷宫试验等方法发现过表达GalT I和 V对脑损伤后神经再生和功能中发挥重要的作用。