AAV-proBDNF对阿尔茨海默病鼠海马DCX阳性细胞数及PSD-95表达的影响

Effects of AAV-proBDNF on hippocampal DCX positive cells and PSD-95 expression in Alzheimer's disease mice

目的探讨侧脑室注射携带抗裂解脑源性神经营养因子前体(proBDNF)基因的腺相关病毒基因整合载体(AAV-proBDNF)对阿尔茨海默病小鼠(AD鼠)海马神经形成与生长的影响。方法取5月龄APP/PS1转基因AD鼠15只,采用抛硬币法随机分为正常对照组(n=5)、空载体对照组(n=5)和AAV-proBDNF组(n=5),AAV-proBDNF组右侧侧脑室注射AAV-proBDNF(4μL,2×10^(13)v.g./mL),空载体对照组右侧侧脑室注射等量空病毒载体,正常对照组右侧侧脑室注射生理盐水(4μL)。于侧脑室注射后4周采用ELISA检测海马proBDNF含量,免疫组化检测海马齿状回DCX阳性细胞数,Western blot检测海马突触后致密物-95 (PSD-95)表达。结果侧脑室注射后4周,AAV-proBDNF组脑内proBDNF表达水平较正常对照组明显增加,且显著高于空载体对照组,差异均具有统计学意义(P<0.05);AAV-proBDNF组海马DCX阳性细胞数为(202.46±24.27),明显低于正常对照组(382.26±37.58,P<0.05)和空载体对照组(365.48±32.68,P<0.05);AAV-proBDNF组海马PSD-95相对含量为(0.62±0.05),明显低于正常对照组(0.82±0.02,P<0.05)和空载体对照组(0.86±0.07,P<0.05)。结论侧脑室注射AAV-proBDNF能够在AD鼠脑内成功表达proBDNF,并可降低AD鼠海马区新生神经元数量及突触可塑性。

Objective To explore the effects of lateral ventricle injection of recombinant adenoassociated virus-brain-derived neurotrophic factor precursor(AAV-proBDNF) on the hippocampal neurogenesis in APPswePS1dE9 transgenic mice(Alzheimer’s disease,AD).Methods Fifteen APP/PS1dE9 transgenic mice(5 months old) were randomly divided into normal control,empty vector control and AAV-proBDNF groups(n=5 in each group).Recombinant vectors of AAV-proBDNF(8×10^10 v.g.) were stereotaxically delivered into the right lateral ventricle in the AAV-proBDNF group,and empty AAV vector and normal saline(4 μL) were given respectively to the mice from the empty vector control and normal control groups.Enzyme-linked immunosorbent assay was conducted to test the proBDNF level in the brain in 4 weeks after injection.The count of doublecortin(DCX) positive cells and the expression of postsynaptic density-95(PSD-95) in the hippocampus were detected by immunohistochemical assay and Western blotting respectively.Results In 4 weeks after injection,the proBDNF level was significantly higher in the AAV-proBDNF group than the normal control group and the empty vector control group(P<0.05).The number of DCX-positive cells in the hippocampus of AAV-proBDNF group(202.46±24.27) was significantly lower than that in the normal control group(382.26±37.58,P<0.05) and empty vector control group(365.48±32.68,P<0.05).In AAV-proBDNF group,the relative content of PSD-95(0.62±0.05) was obviously lower than the normal control group(0.82±0.02,P<0.05) and vector control group(0.86±0.07,P<0.05).Conclusion Lateral ventricle injection of AAV-proBDNF can induce the expression of proBDNF in the brain of AD mice,and reduce the number of new neurons and synaptic plasticity in the hippocampus.