应用原位注射温控型GDNF-肝素-泊洛沙姆水凝胶提高GDNF脊髓损伤治疗的有效性和安全性研究

With the astounding numbers of various accidents in modern society, the incidence of spinal cord injury is increasing, which presents a main threat for young peoples. According to statistics, the average age of patients suffered from spinal cord injury is only 33.4 years old. Since spinal cord injury belongs to a nerve injury disease, traditional chemical treatment can barely protect the survival neurons from secondary injury, without any effects to repair the impaired neurons and axons. Therefore, the sensory and motor ability of patients can not be restored after traditional chemical treatment. Glial cell line-derived neurotrophic factor (GDNF) has a potential for the treatment of spinal cord injury. Its repairing effects on impaired neurons and axons have been confirmed in animal models of spinal cord injury. However, GDNF has a poor physical stability and can not enter the spinal cord tissue under intravenous administration because of the blood-spinal cord barrier. Based on our previous experiments, new hydrogel combined with in situ administration are proved to promote macromoleculars enter the spinal cord tissue. In this project, a novel temperature sensitive hydrogel combined with in situ administration has been used to deliver GDNF into spinal cord tissue. A novel synthetic polymer --" Heparin-poloxamer" (HP), is used as a hydrogel matrix in the hydrogel formulation. HP has a high affinity with GDNF and thus protect GDNF from biological degradation. Experiments are designed to investigate the availability of the in situ GDNF-HP temperature sensitive hydrogel for enhancing GDNF delivery into the spinal cord tissue. Modern pharmacological and pharmacokinetic studies are used to gain a comprehensive evaluation of the efficacy and safety of GDNF-HP temperature sensitive hydrogel on the impaired neurons and axons of spinal cord injury animals. The results of this research will contribute to solve the limitation of modern biotechnological treatment for spinal cord injury.

各类事故造成的脊髓损伤发生率逐年增高，且呈年轻化趋势，患者平均年龄仅33.4岁。由于是神经损伤疾病，脊髓损伤化学药物治疗仅可保护残存神经元免受二次损伤，但无法修复受损神经元及轴突，患者的感觉及运动能力不能恢复。胶质细胞源性神经营养因子（GDNF）具有修复损伤神经元及轴突的双重作用，其效果已在脊髓损伤动物模型上验证。但是GDNF稳定性差，静脉给药难以透过血-脊髓屏障。课题组前期应用水凝胶结合原位给药技术，发现可以促进大分子药物进入脊髓组织。本项目应用自主合成的高分子材料肝素-泊洛沙姆（HP），制备了新型GDNF-HP温敏型水凝胶，利用HP对于GDNF亲和性增加药物稳定性，结合原位注射技术有效递送GDNF进入脊髓组织。通过现代药理学和药动学研究方法，全面评价GDNF-HP温敏型水凝胶经原位注射进入脊髓组织后修复损伤神经及轴突的有效性和安全性，探索克服脊髓损伤等神经受损生物药物治疗瓶颈的新技术。

化学药物治疗脊髓损伤仅能保护残存神经元免受二次损伤，很难促进轴突再生和修复受损神经元。胶质细胞源性神经营养因子（GDNF）修复损伤神经元及轴突的双重作用已得到实验动物的验证，但是GDNF作为一种大分子蛋白药物，其稳定性差，并难以透过血-脊髓屏障，因缺乏高效、缓释的药物递送系统而应用受限。本项目利用既有温度敏感性又保留了肝素较好的抗凝活性的新型高分子材料HP结合夹心冷法制备包载GDNF的肝素-泊洛沙姆温控型水凝胶（GDNF-HP），解决GDNF在脊髓损伤应用的递送问题。GDNF-HP水凝胶胶凝温度为37℃，采用HP浓度为17%的优化处方，具有适宜的力学强度和弹性模量，SEM镜下观察凝胶呈多孔三维网状结构，适宜包载大分子药物GDNF，可以作为原位注射给药的研究。FITC-HP水凝胶明显增大细胞摄取率。原位注射FITC-HP温控型水凝胶的大鼠，FITC明显浓集于脊髓，血药浓度显著下降，具有高效靶向并降低药物全身循环不良反应的作用。GDNF-HP温控型水凝胶在细胞层面有效提高损伤细胞的存活率并修复受损的轴突，减少细胞凋亡。GDNF-HP温控型水凝胶经原位注射给药显著增强GDNF对脊髓损伤修复作用、再生作用，髓鞘排列整齐，无明显脱髓鞘现象，从微观上揭示其修复作用。联合Tunel染色，LC-3、Beclin-1蛋白表达的改变，从分子层面揭示了神经修复效应与自噬抑制凋亡的机制有关。由此得出结论， GDNF-HP水凝胶联合原位注射技术可显著增强GDNF促进脊髓损伤神经再生的作用。HP水凝胶包载GDNF保护其活性的同时还起到骨架支撑作用，结合原位注射给药的方式实现GDNF的局部长效释放，有效提高了GDNF对受损神经组织的保护作用，增强了GDNF在受损脊髓处神经功能重建及神经再生作用的效果，克服了目前GDNF全身给药靶向性低，局部给药药效持续时间短的瓶颈，为脊髓损伤的高效治疗提供一种新的策略。