BDNF及GDNF干预吗啡成瘾行为的基因治疗策略与机制研究

Drug addiction is a chronic and recurrent brain disease whose core problem is abnormal changes in neural plasticity of the midbrain-limbic dopamine system (MLDS). Studies suggested that brain-derived neurotrophic factors (BDNF) and glial-derived neurotrophic factors (GDNF) may play a negative role in morphine addiction. Our previous study has identified that the adeno-associated virus (AAV), carrying the fusion gene of the target protein with the transmembrane peptide named Ant, could serve as an intervention strategy of central nervous system (CNS) disease by nasal administration via the "nasal-brain pathway". On the basis of our previous study, we will construct recombinant AAVs that could secrete BDNF or GDNF with membrane-penetrating effects, and different fluorescent labels will be added to the target protein and virus capsid separately. Then, it would be nasally delivered to normal mice to observe the expression and distribution of the virus capsid and target protein in the nasal mucosa and brain, clarifying the security of the system and brain areas of main effeteness. Next, it will be nasally delivered to morphine addiction mice model to investigate its interventional effects on morphine addiction. And the neuroplasticity mechanisms of the intervention will be explored in the key brain area of MLDS at different level, including structural, efficacious and molecular level. We wish to establish a new system for the intervention of morphine addiction based on the principle and method of gene therapy.

药物成瘾是一种慢性、复发性脑疾病，其核心问题是中脑边缘多巴胺系统（MLDS）的神经可塑性的异常改变。研究提示脑源性神经营养因子（BDNF）及胶质源性神经营养因子（GDNF）在吗啡的成瘾过程中可能起负向调控作用。我们的前期研究表明，携带目的蛋白-穿膜肽融合基因序列的腺相关病毒（AAV），经由“鼻-脑通路”给药方式，可作为中枢神经系统疾病的干预策略。据此，本项目拟构建可分泌表达具有穿膜作用的BDNF或GDNF的重组AAV，并分别在目的蛋白、病毒衣壳上添加不同的荧光标签，首先对正常小鼠经鼻给药，观察病毒衣壳及其分泌表达的蛋白在鼻黏膜及脑内的循行、分布情况，明确体系的安全性、时效性及路径机制；其次经鼻给药考察其对小鼠吗啡成瘾模型的干预效力，并在MLDS重点脑区分别探索干预起效的结构、效能、分子层面的神经可塑性机制，以期建立一种基于基因治疗原理与方法的干预吗啡成瘾行为的全新途径与方法。

药物成瘾是一种药物诱导产生的持久而稳定的中脑多巴胺奖赏系统（VTA-NAc）的适应性/可塑性的异常改变。我们通过综述研究，发现脑核团中脑源性神经营养因子（BDNF）分子水平与吗啡成瘾密切相关。我们进行了动物实验，将 BDNF-AAV注射到小鼠腹侧被盖区（VTA），研究BDNF过表达对吗啡诱导的条件位置偏爱（CPP）模型中不同阶段（形成-消退-点燃）的CPP值的影响及伴随的相关分子变化。结果发现BDNF/TrkB/CREB在慢性吗啡成瘾不同阶段发挥着不同的作用，BDNF是治疗吗啡成瘾的潜在的辅助药物。.同时，我们研究了组氨酸三联体核苷酸结合蛋白1（HINT1）敲除鼠在吗啡成瘾时，多个脑区[额叶皮层(PFC)，伏隔核(NAc)，尾状壳核(CPu)，海马(HIP)]中HINT1分子的变化。主要考察吗啡如何影响成瘾行为（条件位置偏好）的动态过程（CPP分形成-消退-复燃三阶段），进行了多层面（动物层面、分子层面）系统而深入的机制研究，以期为戒除毒瘾和预防复吸的研究提供有益帮助。.