肝素调控PTN/syndecan-3对心肺复苏后脑缺血再灌注损伤和修复机制研究

cerebral injure after cardiac arrest is the main cause of the low survival rate and high the disability rate in patients after resuscitation, which has not been solved nowadays .Axonal regrowth is an important method to solve cerebral injure after resuscitation. We have preliminarily proved that heparin can effectively raise the level of Axonal regrowth of nerve cells, which can significantly reduce cerebral injure after cardiac arrest. PTN is an important factor in the axonal growth of neurons, and the activation of PTN requires the binding of syndecan-3 to its receptor. Both heparin and syndecan-3 core protein bound polysaccharide has a similar glycosaminoglycan structure at the same time. Therefore, this project proposed innovatively that heparin can lead RET protein phosphorylation through PTN-Syndecan Signaling Pathway, and protect nerve cells after injury of cardiac arrest, by timely promoting hypoxic neuronal axon regrowth. This study aimed to explore the relationship between coagulation activation, expression of leukocyte PTN gene and the survival rate of the patients and cerebral function at clinical level,simultaneously, we verified the proposed hypothesis at the animal and cell molecular level, detected the expression and activation of PTN protein, syndecan-3 protein and RET protein and explored the activation mechanism of Axonal regrowth of nerve cells, and,so as to provide theoretical basis that heparin can be uses as a new cardiopulmonary resuscitation medicine.

心脏骤停后脑损伤是造成复苏后患者存活率低和伤残率高的主要原因，目前尚缺乏有效的治疗措施，而神经元轴突再生是解决复苏后神经功能障碍的重要着手点。我们已经初步证实肝素可有效提高神经元再生，显著减轻心跳骤停后脑损伤。PTN是神经元发育阶段轴突生长的重要因子，而PTN的活化需与其受体syndecan-3结合。肝素与syndecan-3核心蛋白结合形成的糖胺聚糖具有类似的多糖结构，因此本项目创新性提出肝素通过PTN- syndecan信号通路，通过激活酪氨酸激酶受体RET，从而调节神经轴突生长，对心脏骤停后脑损伤产生保护作用。本课题拟通过在临床水平探讨患者凝血系统激活、白细胞PTN基因表达与患者生存率及脑功能的关系，同时在整体动物和细胞水平对提出的假说进行验证，检测syndecan-3、PTN及RET蛋白的表达及活化，为把肝素作为新的心肺复苏用药提供理论依据。

心脏骤停后脑损伤是造成复苏后患者存活率低和伤残率高的主要原因，目前尚缺乏有效的治疗措施，而神经元轴突再生是解决复苏后神经功能障碍的重要着手点。临床中发现低凝状态的病人有良好的神经预后，本研究采用在体实验动物和体外细胞实验来验证肝素抗凝是否也能达到此效应。肝素作为临床中常用的抗凝药物，已初步证实它可有效提高神经元再生，显著减轻心跳骤停后脑损伤。为此我们就其可能机制进行研究，PTN作为神经元发育阶段轴突生长的重要因子，其活化需与受体syndecan-3结合，而肝素与syndecan-3侧链的糖胺聚糖具有类似的多糖结构，因此本项目创新性提出肝素通过PTN/syndecan-3信号通路，通过激活酪氨酸激酶受体RET，从而调节神经轴突生长，对心脏骤停后脑损伤产生保护作用。.该项目针对目前心跳骤停-心肺复苏后中枢神经元损伤难以修复的现状，通过临床实验、在体动物实验、体外细胞实验等方面展开研究，采用临床常用药肝素干预，研究其提高自主循环恢复率、改善形态学损伤、保护神经元功能相关机制；利用大鼠窒息性心脏骤停与细胞糖氧剥夺-复氧模型，探讨肝素通过PTN/Syndecan-3信号通路调控神经元修复，达到脑保护的目的。在实验过程中，我们发现肝素处理组神经元生长情况较未用肝素处理组良好，神经元包体相对完整，很少见碎片，轴突少见缩短和贴壁不良现象。提示肝素预处理对原代海马神经元适应低氧环境有一定的保护作用。.采用RT-PCR、Western Blot、免疫荧光、ELISA等实验技术，用肝素干预，与生理盐水组相比，无论是在体内还是体外，肝素预处理组大鼠及细胞的神经元修复因子NF-200和轴突生长因子TAU,信号通路中的关键分子PTN、syndecan-3等都明显增高，证实肝素可在神经细胞损伤以及修复过程中起保护作用，可能通过调控PTN/Syndecan-3通路促进神经元修复，发挥促轴突生长从而保护脑功能。