摘要
目的观察人α-突触核蛋白(α-synuclein,α-Syn)和其突变体A30P和A53T对大鼠原代培养神经元突起生长的影响,明确α-Syn的生理功能,揭示突变体A30P和A53T在帕金森病的发病机制中的作用。方法取大鼠大脑皮质神经元分组培养,在细胞外添加A30P、A53T和α-Syn,培养1 h、2 h和4 h后固定,比较A30P、A53T与α-Syn对神经元突起生长的影响。神经元突起以成像显微镜观察测量,Western blotting法、免疫荧光法、单克隆抗体阻断实验鉴定各蛋白作用的特异性。结果添加α-Syn组的神经元培养至1、2和4 h时,其突起的平均长度大于对照组和添加A30P、A53T组(P<0.05)。A30P、A53T组和对照组的神经元突起长度差异无统计学意义(P>0.05)。增加蛋白的含量,浓度越高,α-Syn组神经元突起的平均长度与添加A30P和A53T组差异越大(P<0.01)。Western blotting和免疫荧光实验明确了外源性α-Syn可以从培养基进入到神经元内,并均匀分布在胞体和突起。而A30P和A53T组,并未发现。结论α-Syn在原代神经元生长初期对其突起生长具有促进作用,突变体A30P和A53T,无促神经元突起生长作用,这一现象可能与其在帕金森病发病机制中的作用有关。
Objective To investigate the effect of A53T and A30P on the neurite outgrowth of brain neurons and indicate its function, explore the mechanism of Parkinson＇s disease. Methods Neurons isolated from the neoeortex of newborn Wistar rats were cultured and corresponding proteins were added into the culture medium to observe the effect of the protein on the neurite outgrowth of the neurons. A53T, A30P and α-synuclein were added into the culture system of neural cells, and the different promoting effect of neufite outgrowth was compared with that of control groups. Western blotting assays and immunofluorescence staining assay were used to confirm whether the result is special. Inverted phase contrast microscope, and photographs were applied to analyze the results with software. Results The mean neurite length of the neurons treated with α-Syn was significantly longer than that of A53T and A30P group neurons（ P 〈 0.05 ）. There was no significant difference between A53T and A30P groups with control group（ P 〉 0.05 ）. Increased concentration of α-synuelein could enhance this effect. The result of Western blotting and immunofluoreseence staining assay showed that α-synuclein could enter the neural cell and enhance the neurite outgrowth. Blockade with monoclonal antibody 3D5 showed that the blocking of α-Syn could significantly inhibit the effect （ P 〉 0.05 ）. Conclusion α-Synuclein can enhance neurite outgrowth of primary cultured neuron in a dosedependent manner. But its mutants A53T and A30P did not show such effect.
出处
《首都医科大学学报》
CAS
2012年第5期629-633,共5页
Journal of Capital Medical University
基金
国家自然科学基金项目(30271437
30270482)
北京市自然科学基金项目(7022011)
吉林省教育厅"十二五"科学技术研究项目
吉教科合字[(2012)-137]~~
关键词
突触核蛋白
原代神经元培养
突起生长
帕金森病
α-synuclein
primary neuronal
culture neurite outgrowth
Parkinson＇s disease
