pBDNF过表达对转基因阿尔茨海默病鼠学习记忆功能的影响

Exogenetic over-expression of pBDNF affects learning and memory in APPswePS1dE9 transgenic mice

目的探讨海马注射携带脑源性神经营养因子前肽(pBDNF)基因的腺相关病毒基因整合载体(AAV-pBDNF)对阿尔茨海默病鼠(AD鼠)学习记忆功能的影响及可能机制。方法选用18只6月龄APP/PS1转基因AD小鼠,采用抛硬币法随机分为空载体对照组、AAV-pBDNF组、AAV-pBDNF+p75NTR抗体组,每组6只。空载体对照组右侧海马注射AAV-GFP(2μL,1×1012 vg/mL),AAV-pBDNF组右侧海马注射AAV-pBDNF(2μL,1×1012 vg/mL),AAV-pBDNF+p75NTR抗体组右侧海马注射AAV-pBDNF(2μL,1×1012 vg/mL)和p75NTR抗体(2μL,10μg/mL)。海马注射4周后行Morris水迷宫实验检测AD鼠学习记忆能力的变化。免疫荧光检测AAV-pBDNF在AD鼠脑内转染情况。Western blot检测海马突触后致密蛋白-95(postsynaptic density protein 95, PSD-95)的表达。结果水迷宫实验结果显示:各组小鼠的运动速度无明显差异。第5天时,对照组与AAV-pBDNF+p75NTR抗体组小鼠的逃避潜伏期均较第1天有明显下降(P<0.05),而AAV-pBDNF组AD鼠的逃避潜伏期虽然也较第1天有所缩短,但差异无统计学意义。AAV-pBDNF组AD小鼠穿越平台次数较其余两组少(P<0.05)。免疫荧光检测结果发现:AAV-pBDNF转染之后,在脑内神经元成功表达pBDNF,而胶质细胞不表达pBDNF。Western blot结果显示:AAV-pBDNF组AD鼠的PSD-95表达量低于对照组和AAV-pBDNF+p75NTR抗体组(P<0.05)。结论 pBDNF可能通过p75NTR受体信号通路下调海马PSD-95的表达,降低AD鼠的学习记忆能力。

Objective To explore the effect and possible machanism of recombinant adeno-associated virus(rAAV) vector containing brain-derived neurotrophic factor propeptide(AAV-pBDNF) on learning and memory in APPswePS1 dE9 transgenic mice(Alzheimer’s disease, AD). Methods A total of 18 APPswePS1 dE9 transgenic mice were randomly and equally divided into control group, AAV-pBDNF group and AAV-pBDNF+p75 NTR antibody group. Recombinant AAV-pBDNF vector(2 μL, 1×1012 vg/mL) were stereotaxically injected into the right side of the hippocampus in AAV-pBDNF group, AAV-GFP(2 μL, 1×1012 vg/mL) into that of the control group, and same amount of AAV-pBDNF and p75 NTR antibody(2 μL, 10 μg/mL) into that of the AAV-pBDNF+p75 NTR antibody group. There were 6 mice in each group. Morris water maze test was performed in 4 weeks after hippocampal injection to detect the changes of learning and memory abilities in AD rats. Immunofluorescence assay was used to observe the transfection ofAAV-pBDNF in the brain. Western blotting was employed to measure the expression of postsynaptic density protein 95(PSD-95) in the hippocampus. Results The results of Morris water maze showed that no significant difference was seen in the movement speed of each group(P>0.05);the escape incubation period on day 5 in the control group and AAV-pBDNF+p75 NTR antibody group was significantly shorter than that of day 1(P<0.05);similar result was seen in the pBDNF group though no statistical difference(P>0.05);and less platforms were crossed in the AD mice of the pBDNF group than those in the other 2 groups on day 6 in space exploration experiment(P<0.05). Immunofluorescence assay showed that after transfection, pBDNF was expressed in the neurons, but not glial cells in the hippocampus. Western blotting results indicated that the expression of PSD-95 was obviously lower in the AAV-pBDNF group than the other 2 groups(P<0.05). Conclusion pBDNF inhibits the learning and memory abilities of AD mice by down-regulating hippocampal expression of PSD-95.