基于Insulin/IGF-1信号通路探讨健脾益智法促进MCAO大鼠神经干细胞增殖分化的机制研究

Stroke is a common ,frequently-occurring disease in clinical . The proliferation, migration and differentiation of endogenous NSCs on damaged brain regions can induce nerve regeneration and repair damaged brain tissue after cerebral ischemia.Many explorations in terms of NSCs proliferation have been made by traditional Chinese medicine, but the mechanism is not clear. Our preliminary research show that Jianpi Yizhi method can contribute to the proliferation of NSCs in cerebral ischemia.The spleen control transportation and transformation. That is to say the spleen governs digestion and absorption of food which related to the energy.Some article suggested that energy metabolism is correlated with NSCs proliferation.Theregore,this topic put forward the hypothesis that Jianpi Yizhi method regulate neural stem cells by controlling energy metabolism.Central to the body’s ability to coordinate energy intake and expenditure is the insulin/IGF-1 signaling pathway. This study take NSCs and MCAO rats as the research carrier, use immunohistochemistry ,PCR,Western Blot and in situ hybridization technique, observe the affects of Jianpi Yizhi method to Insulin/IGF-1 signaling pathway from in vivo and in vitro, explore the molecular mechanism of regulating the NSCs proliferation, attempt to reveal the relationship between the effect of Jianpi Yizhi method and the nerve regeneration from micro level, which may provides new thinking and methods for prevention and treatment of ischemic stroke in clinical.

脑卒中是临床常见病、多发病。脑缺血后受损脑区内源性NSCs的增殖、迁移和分化可诱导神经再生，修复受损脑组织。中医在NSCs增殖分化方面进行了许多探索，但对机制缺乏清晰的认识。本课题组前期研究结果表明健脾益智法可以促进脑缺血NSCs的增殖。“脾主运化”，主饮食物的消化与吸收，与能量相关。相关文献表明，能量代谢与NSCs增殖分化有相关性。因此，本课题提出假说，认为健脾益智法是从能量代谢的角度对神经干细胞进行调控的。胰岛素/胰岛素样生长因子1信号通路是身体协调能量摄入与支出的中心。因此，本课题以NSCs及MCAO大鼠为研究载体，采用免疫组化、PCR、Western Blot、原位杂交等技术，从体内及体外观察健脾益智法对Insulin/IGF-1信号通路的影响，探讨调控NSCs增殖分化的分子机制，尝试从微观水平揭示脾的“运化”作用与脑神经再生的关系，为临床防治缺血性脑卒中提供新的思路与方法。

“脾主运化”，主饮食物的消化与吸收，与能量代谢有关。相关文献表明，能量代谢与神经干细胞的增殖分化有相关性。胰岛素/胰岛素样生长因子1信号通路是身体协调能量摄入与支出的中心，与神经干细胞的增殖分化相关，本课题以大脑中动脉栓塞大鼠为研究对象，以胰岛素/胰岛素样生长因子1信号通路为切入点，从体内、体外实验观察健脾益智法对胰岛素/胰岛素样生长因子1信号通路的影响，探讨健脾益智法调控神经干细胞增殖分化的机制，发现：体内实验（1）健脾益智胶囊可减少大鼠脑缺损体积，抑制通路后，脑梗死体积增大。（2）健脾益智胶囊可促进神经干细胞增殖，分化为星形胶质细胞，未见分化为神经元，可能与观测时间较短有关。（3）健脾益智胶囊可改变术后大鼠血清胰岛素水平，术后1d抑制通路后，血清胰岛素水平明显降低。（4）健脾益智胶囊能够促进PI3K、AKT基因及p-Akt、p-mTOR、p-FoxO、p-P70S6K、p-4E-BP1蛋白的表达，抑制 PTEN基因、p-PTEN蛋白的表达。抑制通路后PI3K、AKT基因及p-Akt、p-mTOR、p-FoxO、p-P70S6K、p-4E-BP1蛋白的表达减少，PTEN基因、p-PTEN蛋白的表达增加。体外实验：（1）健脾益智胶囊促进神经干细胞增殖的最适浓度为0.5mg/ml，通路移植后增殖明显减少。（2）经鉴定，课题所取材的神经干细胞可增殖并分化为神经元、星形胶质细胞、少突胶质细胞，为神经干细胞无误。（3）健脾益智胶囊能够促进神经干细胞PI3K、AKT基因及p-Akt、p-mTOR、p-FoxO、p-P70S6K、p-4E-BP1蛋白的表达，抑制 PTEN基因、p-PTEN蛋白的表达。抑制通路后PI3K、AKT基因及p-Akt、p-mTOR、p-FoxO、p-P70S6K、p-4E-BP1蛋白的表达减少，PTEN基因、p-PTEN蛋白的表达增加。