摘要
目的构建及评价收集特定部位的标记神经元,对这类神经元的微量蛋白水平采用定量的方法,并将此方法初步应用于小鼠颅脑创伤后海马亚区神经元中。方法硫堇染色未固定处理的冰冻切片脑组织神经元,激光显微切割收集海马CA1区和DG区神经元,全自动毛细管Western blot检测收集细胞的磷酸化蛋白激酶A(p-PKA)蛋白水平。结果全自动毛细管Western blot对低浓度蛋白的检测下限低于传统Western blot 1个数量级,其线性相关系数为98. 85%,日内和日间精密度分别为2. 50%和4. 09%。本方法应用于颅脑创伤小鼠脑组织,发现伤后1 d海马DG区神经元p-PKA显著增高(P <0. 01),CA1区无变化。结论本方法基于激光显微切割和全自动毛细管Western blot,能够实现精确收集指定神经元并准确定量微量蛋白,成功应用于目标神经元蛋白定量,具有在细胞类型众多且混杂分布的组织中进行细胞和分子水平研究的应用前景。
Objective To establish a quantitative detection system for phosphorylated protein kinase A （p-PKA） in labeled hippocampal neurons of mice following mild brain trauma using laser microdissection combined with automated capillary electrophoresis Western blotting. Methods From a mouse model of mild traumatic brain injury, fresh samples of the hippocampal tissues were collected for preparing frozen sections with Thionine staining. Laser microdissection was used for collecting the neurons in the hippocampal CA1 and DG regions and the expression level of p-PKA was detected using fully automated capillary Western blotting. Results The lower detection limit of automated capillary electrophoresis Western blotting for low-concentration proteins was lower than that of conventional Western blotting by 1 order of magnitude. This combined detection system had a linear correlation coefficient of 98.85% for p-PKA detection with inter-and intra-day precisions （RSD） of 2.50% and 4.09%, respectively. In the mouse model with traumatic brain injury, the level of p-PKA in the hippocampal DG region increased significantly at 1 day after the injury （P〈0.01）, but its expression showed no significant changes in the CA1 region. Conclusion Laser microdissection combined with automatic capillary Western blotting allows collection of specific neuronal cells and accurate quantification of low-concentration proteins, and can be potentially used for cellular and molecular studies of tissues with complex distribution of different cell types.
作者
杨楠
谭思维
彭艳
周元国
宁亚蕾
YANG Nan;TAN Siwei;PENG Yan;ZHOU Yuanguo;NING Yalei(State Key Laboratory of Trauma,Burn,and Combined Injury,Molecular Biology Center,Collaborative Innovation Center of Brain Science,Institute of Surgery Research,Third Affiliated Hospital,Army Medical University（Third Military Medical University）,Chongqing,400042,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第20期1846-1850,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81571888)
重庆市基础与前沿研究计划项目(CSTC2014jcyjA10072)
