脑缺血后强制性运动疗法促进海马新生神经细胞成熟及存活的机制研究

Stromal cell-derived factor 1(SDF-1) and its proteolytic product, SDF-1(5-67) coexist in the same local microenvironment. SDF-1 regulates cAMP and CREB activation through GABA-mediated excitation, enhances the survival of newborn neurons in dentate gyrus and promotes their dendritic development. SDF-1(5-67) that inhibits cAMP-CREB signaling pathway through Gi/Go coupled receptors has the opposite effect on neurogenesis. Our preliminary data suggest that constraint-induced movement therapy (CIMT) may affect the downstream cAMP-CREB signal by regulating SDF-1 and SDF-1(5-67) pathways. Therefore, CIMT may reverse the local microenvironment which is harmful to the maturation and long-term survival of newborn neurons in dentate gyrus after stroke. We will employ in vivo retroviral strategy combined with shRNA technology to manipulate each molecule of the SDF-1→GABA excitation→cAMP/CREB and SDF-1(5-67)→Gi/Go→cAMP/CREB signaling pathways exclusively in newborn neurons of dentate gyrus to verify the hypothesis from a new interdisciplinary perspective. The study may provide a new target for stroke treatment.

脑缺血后间质细胞源性因子-1(SDF-1)及其蛋白裂解产物SDF-1(5-67)共存于一个局部微环境之中。SDF-1通过γ-氨基丁酸(GABA)介导的突触传入来激活环磷酸腺苷(cAMP)及下游的cAMP 应答元件结合蛋白(CREB)信号通路，从而促进海马新生神经细胞的存活及树突发育。SDF-1(5-67)通过Gi/Go蛋白抑制cAMP-CREB通路，对神经发生起到相反的作用。 我们的前期工作提示，强制性运动疗法可能通过调控SDF-1及SDF-1(5-67)相关通路的功能，从而逆转了不利于脑缺血后新生神经细胞成熟及存活的局部微环境。我们拟应用在体逆转录病毒转染及shRNA干扰技术来选择性调控海马新生神经细胞的SDF-1→GABA突触传入→cAMP/CREB及SDF-1(5-67)→ Gi/Go→cAMP/CREB通路的各个环节，从多角度多层次来验证这一假设。这将为脑梗死的治疗提供新的靶点。

神经再生是脑缺血后功能恢复的结构基础，康复训练能够影响神经再生。CIMT广泛地应用于康复领域，并被证实能够有效地促进肢体功能恢复。但是，CIMT对脑缺血后神经再生的作用及机制尚不清楚。本课题应用转基因技术及细胞、神经纤维示踪标记方法并结合行为学测试来探讨脑缺血后CIMT对神经发生，皮质脊髓束轴突再生及海马区颗粒细胞和树突再生的影响，以及在此过程中CIMT发挥作用的机制和通路。结果发现：1. 脑缺血后CIMT能够促进患侧肢体运动功能恢复。2.CIMT能够促进脑缺血后大鼠新生神经细胞的再生和存活，CIMT能够促进脑缺血后健侧的皮质脊髓束向除神经支配的对侧脊髓灰质芽生及形成分支，以及增加海马体中新生颗粒细胞的数量和树突状复杂性。3. CIMT能够提高脑缺血后大鼠皮层中SDF-1, cAMP, PKA 及磷酸化CREB的水平。4应用PKA的拮抗剂H89拮抗cAMP/PKA/CREB信号转导通路后，CIMT不能发挥其有益作用。提示CIMT通过cAMP/PKA/CREB信号转导通路发挥其促进神经系统可塑性及功能恢复的作用。这些结果从细胞分子水平阐明了脑缺血后CIMT对神经再生的作用及可能的机制，为脑梗死的临床康复提供了理论基础。