利用SOCS3抑制大鼠脑缺血后反应性星形胶质细胞增生促进神经损伤修复的作用及机理研究

Reactive astrogliosis, characterized by cellular hypertrophy, hyperplasia, proliferation and scar formation, plays a major role in delayed degenerative processes of ischemia by impairing the axon regeneration and functional recovery after ischemic brain injury. Therefore, suppressing the reactive astrogliosis might ameliorate the brain damage and enhance the functional recovery after ischemic brain injury. .Suppressors of cytokine signaling (SOCS), a family of genes involved in inhibiting the JAK/STAT signaling pathway, play a broad spectrum of roles in both the maintenance of homeostasis and the development of some diseases because JAK/STAT pathway is involved in cell proliferation, differentiation and transformation. Therefore, SOCS is considered to be a potential therapeutic target in many human diseases including central nervous system (CNS) disorders. It has been reported that infusion of antisense SOCS3 into the ventricle before middle cerebral artery occlusion (MCAO) increases the lesion volumes and worsens the neurological outcomes, suggesting that SOCS3 is neuroprotective in the brain after ischemic stroke. However, the underlying mechanism for this effect has not yet been elucidated. .Here we raise a hypothesis that the protective effect of SOCS3 on ischemic stroke is due to the inhibition of JAK/STAT pathway within the astrocytes and thereby attenuating the reactive astrogliosis. In order to validate this hypothesis, we plan to perform experiments as follows: (1) First, a modified rat neural stem cell (NSC) subline with stable and long-term ectopic expression of SOCS3 (NSC-SOCS3) will be constructed by Lentivirus-mediated transduction; (2) Then, the morphology and the bio-behaviors such as proliferation and viability of the in vitro cultured rat astrocytes will be observed after being co-cultured with NSC-SOCS3 or being cultured in NSC-SOCS3-conditioned medium. (3) Later, the influence of SOCS3 on axon extension and astrogliosis in the ex vivo organotypic rat brain slice cultures under normoxic and hypoxic conditions will be observed after NSC-SOCS3 transplantation. (4) After that, the lesion volumes and astrogliosis in the rat brains and the rat neurological outcomes after NSC-SOCS3 transplantation will be compared with those in the control groups to evaluate the functional recovery-accelerating efficiency of SOCS3. (5) Finally, in order to further examine its signaling pathways, the level of phosphate STAT3 will be determined before/after treatment in cell cultures and organotypic slice cultures, and the effect of SOCS3 on astrocytes as previous described will be re-checked after using AICAR to specifically activate JAK/STAT3 pathway. The implementation of this project will clarify the role of SOCS3 in ischemic stroke, elucidate its underlying mechanism(s) and further highlight the potential clinical importance of SOCS3 in CNS diseases.

星形胶质细胞反应性增生可加重迟发性脑缺血损伤，并阻碍损伤修复和神经功能重建。细胞因子信号途径抑制蛋白SOCS3对脑缺血有保护作用，是否与其抑制星形胶质细胞反应性增生有关，目前并不清楚。本研究拟构建稳定表达SOCS3的工程化神经干细胞NSC-SOCS3，分别进行SOCS3工程化细胞移植、体外共培养或以工程化细胞条件培养基处理，通过观察脑梗死体积、神经突触数量、突起长度及动物行为学评分，观察星形胶质细胞数量、增殖、形态及功能变化，分别在动物个体、器官型脑组织片及细胞水平阐明SOCS3抑制缺血缺氧损伤后星形胶质细胞反应性增生、促进脑功能恢复的作用；通过检测星形胶质细胞内磷酸化STAT3水平及特异性激活JAK/STAT3信号对SOCS3作用的影响，分析其作用的信号转导机制，探索利用SOCS3提高脑缺血治疗效果、促进损伤修复和神经功能重建的价值和机制，为开发脑缺血临床治疗手段奠定理论和实验基础。

星形胶质细胞反应性增生在脑损伤修复过程中发挥重要的意义，项目围绕利用SOCS3抑制JAK-STAT3信号通路，从而影响星形胶质细胞反应性增生展开。首先设计合成由穿膜肽TAT与SOCS3氨基端激酶抑制区KIR组成的小分子融合肽，在证实融合肽TAT-KIR具有有效地跨膜转运及抑制JAK-ATAT3磷酸化的基础上，分别构建体外胶质细胞反应性增生（IL-6刺激培养的星形胶质细胞、器官型脑组织片损伤）模型以及体内胶质细胞反应性增生（创伤性脑损伤）模型，通过培养基添加或脑损伤局部注射TAT-KIR完成对其星形胶质细胞的影响。结果证实：①体外条件下，TAT-KIR可有效抑制IL-6刺激引起的星形胶质细胞存活与增殖，对活化后星形胶质细胞形态特征和基因表达改变也有一定的逆转作用。②体外条件下，TAT-KIR显著抑制星形胶质细胞生成，抑制GFAP蛋白合成，抑制神经干细胞/前体细胞向星形胶质细胞方向分化。③体外条件下，TAT-KIR显著抑制脑组织机械性损伤后星形胶质细胞反应性增生，细胞数量显著减少，脑组织GFAP蛋白含量显著下降。④在体条件下，TAT-KIR有效抑制创伤性脑损伤后损伤局部反应性胶质细胞增生，减轻星形胶质细胞活化，但是仍保留部分活化的星形胶质细胞，为其发挥保护性作用提供基础。⑤在体条件下，TAT-KIR抑制损伤局部星形胶质细胞反应性增生同时，显著抑制全脑小胶质细胞活化。与此同时，神经功能恢复加速，但对神经元损伤修复的影响没有显著性变化。以上研究结果提示用小分子融合肽TAT-KIR有望成为有效改善脑损伤后星形胶质细胞反应性增生、促进神经功能恢复重要手段，具有极高的应用价值。有关最佳使用途径及使用时间窗，仍需要更为深入的研究。