柔性纳米脂质体介导脑源性神经营养因子经嗅区入脑发挥帕金森病模型大鼠多巴胺神经元保护作用实验研究

Parkinson's disease (PD) is a chronic, neurodegenerative disease and usually needs expensive surgery. Though chemical drug substitution therapy can improve symptoms, it fails in preventing dopamine neuron degeneration. As a neurotrophic factor, brain derived neurophic factor (BDNF) has a p potential for the treatment of Parkinson's disease. Based on many reportes, BDNF has the potentials to repair the damaged substantia nigra - striatal system and reverse the motor neuron defects in animal models of Parkinson's disease. However, it is difficult for BDNF to enter the central nervous system because of the numerous protective barriers in brain. Olfactory area olfactory nerve has a direct contact with the central nervous system. Through olfactory nerve pathway, we have successfully delivered fluorescent protein into the brain by self-made flexible liposomes. Combined with active loading technique, a novel flexible nano-liposome with colloidal core structure will be used to prepare BDNF flexible nano-liposome. BDNF flexible nano-liposomes are delivered into the brain using the olfactory pathway. With modern pharmacology method, following will be studied including the distribution and elimination of BDNF in the brain, nasal and brain tissue toxicity, establishment of Parkinson's disease animal model and evaluation of the protective effect of dopamine neurons of BDNF. Combining the advantages of flexible nano-liposome delivery technology and olfactory nerve pathway into brain, the practicality and security of BDNF flexible nano-liposomes will be fully investigated by modern pharmacological research methods in order to provide technical solutions for neurophic factor drugs in PD treatment. The results of this research will contribute to enhance the level of biological drug treatment for Parkinson's disease.

帕金森病是中枢神经系统变性疾病，化学药物替代疗法只能改善症状，不能阻止多巴胺能神经元进行性变性。脑源性神经营养因子(BDNF)对受损的黒质-纹状体系统有显著的修复作用，可在帕金森病动物模型上逆转运动神经元缺陷，具有治疗帕金森病潜力，但BDNF传统给药方式因血脑屏障阻碍难以进入脑内起效。鼻腔嗅区通过嗅神经与中枢神经系统直接联系，课题组利用柔性纳米脂质体结合鼻嗅区入脑通路技术成功将荧光蛋白直接递送入脑。本项目首次采用创新的HP胶体核心柔性脂质体技术结合pH梯度主动载药技术，制备BDNF柔性纳米脂质体，利用鼻嗅区通路结合柔性纳米脂质体技术递送BDNF入脑，通过现代药理学方法研究BDNF鼻嗅区通路入脑后的药物分布和消除情况、鼻黏膜和脑组织毒性、脑内蓄积等；采用多种评价方法进行BDNF入脑后对帕金森病模型大鼠多巴胺神经元保护作用评价。本研究成果将有力提升我国在帕金森病等顽固性脑病的生物药物治疗水平。

脑源性神经营养因子(BDNF)对受损的黒质-纹状体系统有显著的修复作用，可逆转帕金森病模型动物的神经元退行性病变，具有治疗帕金森病(PD)的潜力。柔性纳米脂质体(FNL)具有穿透能力强、毒性低、靶向性好、稳定性高等优势，是药物靶向递送的良好载体。经鼻嗅区给予包载药物的柔性纳米脂质体后，药物可绕开血脑屏障直接递送进入中枢神经系统，大大提高脑内药物浓度，对脑部退行性疾病具有良好的治疗效果。本项目通过W/W乳化法结合冷冻干燥技术制备外观圆整规则、粒径在200 nm左右且分布集中、包封率高、稳定性好的BDNF柔性纳米脂质体(BDNF-FNL)，并对柔性纳米脂质体介导BDNF经鼻嗅区入脑安全性及初步药动学进行研究，然后对帕金森模型大鼠连续14天经鼻嗅区给予BDNF柔性纳米脂质体治疗，以阿扑吗啡诱导实验评价制剂对帕金森模型大鼠的行为学改善情况，用HE染色法考察制剂对黑质区微观结构的影响，应用高效液相-电化学检测技术(HPLC-ECD)检测大鼠纹状体内单胺类神经递质的变化，通过免疫组化染色法检测黑质内酪氨酸羟化酶(TH)含量的变化，综合评价经鼻给予BDNF-FNL对帕金森模型大鼠的疗效。研究结果表明，与帕金森模型组大鼠相比，经鼻给予制剂组(BDNF-FNL/in组)大鼠的行为学显著改善(P<0.01)，大脑损伤侧的细胞数明显增多且神经胶质细胞增生、炎性细胞浸润和结缔组织增生的情况明显改善，纹状体内单胺类神经递质的含量明显升高，且损伤侧黑质内的TH含量显著上升(P<0.05)。由此得出结论，本项目创新制备的BDNF柔性纳米脂质体经鼻嗅区给予后，可绕过血脑屏障直接递送BDNF进入中枢神经系统，发挥多巴胺能神经元保护作用，对帕金森模型大鼠呈现较好的疗效。本项目成果为帕金森病的生物药物高效治疗提供新的思路。