Cystatin C对脑缺血后海马神经发生的影响及机制

With the increased incidence of ischemia, more and more people are suffered from vascular dementia (VD). There is still no effective method for clinical therapy on VD, as its unclear mechanism. Previous studies have proved that the neurogenesis in hippocampus has played a role in the formation process of cognitions. Usually, the neurogenesis in hippocampus becomes active after ischemia, some study showed that most of the neural stem cell differentiated into glial cell after ischemia, which may damage the neuron. The change of neurogenesis after ischemia may affect on the patient's cognitions. The main regulation factors about neurogenesis and their related mechanisms still needed to be studied. It would be benefit to the clinical therapy on VD when the main regulation factors about neurogenesis and their related mechanisms are clarified. Cystatin C(Cys C) is targeted many pathological physiology process of neurological disease, but its definite function is still unclear. Previous study showed the expression level of Cys C was significant increase in hippocampal region after ischemia. Cys C is inclined to formation oligomer, which might lead to different effects of Cys C on neural cell. We have proved that Cys C played a role in neuronal apoptosis and induced the tyrosine hydroxylase gene expression in differentiated neuronal cell through JNK pathway. The oligomer of Cys C has been detected in the serum of the ischemic patient in our study. We hypothesized that the effect of Cys C on the process of hippocampal neurogenesis after ischemia may be different between Cys C monomer and Cys C oligomer. In this study, we want to study the change of the neurogenesis process in the hippocampus after ischemia and the involvement of Cys C monomer and Cys C oligomer in the neurogenesis process by an established ischemic mouse model and cell culture system. We will evaluate the neurogenesis change through examining the proliferation , migration and differentiation of the neural stem cell using Brd U staining, immuno-histochemical staining, laser capture microdessection, Real Time PCR and Western blotting.We aim to provide an useful basic theory for the clinical therapy on VD.

血管性痴呆发病率逐年升高，临床仍无有效治疗方法。脑缺血后，海马区Cystatin C（Cys C）表达水平增高，Cys C已被发现参与神经发生过程，但其功能仍不清楚。前期研究中发现，脑缺血患者血清中存在Cys C寡聚体，Cys C寡聚体对脑缺血后海马神经发生的功能及机制研究较少。本项目拟利用小鼠慢性脑缺血模型，采用组织学及分子生物学技术观察脑缺血后Cys C在海马区表达特点，并结合脑缺血后海马区神经发生的变化，探讨脑缺血后CysC对海马区神经发生的影响；利用体外海马神经干细胞培养体系，观察不同条件下，不同结构形式的Cys C对海马神经干细胞的增殖能力，迁移及分化过程的影响；通过在培养体系中添加MAPK通路抑制剂，观察Cys C对神经发生影响的变化，探讨Cys C通过MAPK通路影响神经发生的机制。探明脑缺血后Cys C对神经发生的影响和机制，将为临床寻找治疗血管性痴呆提供一种新的思路。

脑卒中的死亡率和发病率极高，随着其发病率的持续增长，血管性痴呆（Vascular dementia ，VD）可能会成为我国老年期痴呆的第一大病因。据相关统计，我国老年痴呆性疾病中VD 的发病率居于第2位。已有研究证明，海马区的神经发生参与认知能力的形成过程。脑缺血改变了海马区的神经发生，但其机制不清楚，目前的相关研究也较少。探明脑缺血后神经发生的调控因素及机制将会推动临床寻找治疗老年期痴呆的有效方案。本项目利用脑缺血模型及海马区神经干细胞培养体系，采用免疫组织（细胞）化学（荧光）染色、Real-time PCR、免疫沉淀及Western blotting等方法，探讨了缺血后海马区神经发生；不同结构形式（单体，寡聚体）的Cys C对脑缺血后海马区神经发生过程/神经干细胞的增殖能力，迁移及分化过程的影响及可能机制。并探讨了临床脑卒中患者血清中Cys C的结构形式。结果发现，脑缺血改变了海马区的神经发生，脑缺血激活了海马DG 区神经干细胞，使干细胞增殖，并向神经元、星形胶质细胞、少突胶质细胞分化，同时逐渐向CA1 区迁移；脑缺血后海马DG 区和 CA1 区 Cystatin C 的表达增高，脑缺血后，海马区Cystatin C寡聚体形成增加。体外细胞模型结果表明，氧糖剥夺导致神经干细胞发生凋亡，增殖能力下降，并使分化后的神经元及神经胶质细胞增殖能力下降；cystatin C影响了脑缺血后海马区神经发生的具体过程。本研究结果必将对临床寻找治疗血管性痴呆的方法提供有力的帮助。