鱼藤酮PLGA纳米粒的制备及诱导大鼠帕金森病模型的实验研究

Preparation of rotenone PLGA nanoparticles and Parkinson's disease model induced in rats

目的:制备鱼藤酮聚合物纳米分散液,建立与人类帕金森病发病特征相类似的动物模型。方法:采用溶剂扩散法制备鱼藤酮聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]纳米粒,检测冷冻干燥保护剂(海藻糖、甘露醇、葡萄糖)及其浓度对冻干样品粒径的影响。将PLGA纳米粒制成冻干粉,28只SD大鼠分为4组,分别为空白对照组(6只),鱼藤酮组(6只),高剂量鱼藤酮PLGA纳米粒组(R-PLGA,8只)以及低剂量R-PLGA组(8只)。各组均为皮下注射给药,空白对照组给予生理盐水,鱼藤酮组给予普通鱼藤酮溶液,R-PLGA组给予鱼藤酮纳米粒冻干粉。除高剂量组每4天给药1次(剂量为3.5 mg/kg)外,其他组均为每3天给药1次,每次剂量均为2.5 mg/kg。各组整个处理过程均为27 d。采用纳米粒度仪/Zeta电位仪测定纳米粒的粒径与Zeta电位;差示扫描量热仪(DSC)分析纳米粒中鱼藤酮的熔点,HPLC法测定鱼藤酮含量。观察大鼠外观行为表现(评分)、体质量、实格试验中移动潜伏期,用免疫组化法测定大鼠黑质酪氨酸羟化酶(TH)免疫阳性细胞数。结果:制得的R-PLGA纳米粒平均粒径为306.5 nm,Zeta电位为-2.66 m V,包封率为89.14%,载药量为18.22%。2%海藻糖作为冷冻干燥保护剂保护作用最好,冻干粉平均粒径312.6 nm。与对照组比较,普通鱼藤酮组与高、低剂量R-PLGA组的大鼠行为学评分升高(P<0.01),移动潜伏期从(0.93±0.21)s延长至(7.35±3.27)s,差异有统计学意义(P<0.05)。对照组TH免疫阳性细胞数为82.06±10.20,低剂量模型组(17.54±4.20)明显减少(P<0.01)。结论:制得可用于制作帕金森病大鼠模型的R-PLGA纳米粒冻干粉。

Objective ： To establish a method for preparing animal models with onset characteristics simi- lar to that of human Parkinson＇s disease（PD）. Methods： The rotenone poly （lactie-eo-glyeolic acid） （PL- CA） nanoparticles was prepared by using solvent diffusion method. Inspecting the types of freeze-drying pro- tectants and concentration on the influence of sample diameter of freeze-drying powder. The Parkinson＇s dis- ease model was induced in adult SD rats by subcutaneous injection of rotenone PLGA,the model of low dose 2.5 mg/kg every 3 days, and high dose 3.5 mg/kg every 4 days. Altogether the drug treatment lasted for 27 days. Diameter and Zeta potential of nanoparticles was determined by nanometer particle size analyzer. With differential scanning calorimetry （DSC）the physical state of rotenone in nanoparticles was analyzed. Rote- none of the contents was determined by HPLC. Grid experiment test were applied to investigate animal be- havior change and prolonged start latency before and after rotenone was exposed. The injury of dopaminergic neuron was examined by the method of immunohistochemisty. Results： The mean diameter of the particle was 306.5 nm and Zeta potention was - 2.66 inV. Its entrapement ratio reached 89.14% with the drugloading of 18.22%. The effectiveness of protection is best when using 2% concentration trehalose. The mean diameter of the particle was 312.6 nm. Compared with the control group, the rats of general rotenone group and model group , behavior score was rised（P 〈 0.01 ） ;the start latency was prolonged to （7.35± 3.27）s （P 〈 0.05 ）, respectively, compared with in control group （ 0.93 ± 0.21 ） s. The number of nigral tyrosine hydroxylase（TH） positive cells decreased significantly from 82.06 ±10.2 in control group to 17.54 ±4.2 （P 〈 0.01 ）. Conclusion： Good quality freeze-dried preparations can be obtained and the adult rat PD models were established successfully under the condition.