[1]杨楠,谭思维,彭艳,等.显微切割联合全自动Western blot在小鼠大脑标记神经元微量蛋白定量中的应用[J].第三军医大学学报,2018,40(20):1846-1850.
 YANG Nan,TAN Siwei,PENG Yan,et al.Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma[J].J Third Mil Med Univ,2018,40(20):1846-1850.
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显微切割联合全自动Western blot在小鼠大脑标记神经元微量蛋白定量中的应用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第20期
页码:
1846-1850
栏目:
基础医学
出版日期:
2018-10-30

文章信息/Info

Title:
Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma
作者:
杨楠谭思维彭艳周元国宁亚蕾
陆军军医大学(第三军医大学)第三附属医院(野战外科研究所)分子生物学中心,国家创伤、烧伤与复合伤重点实验室,陆军军医大学脑科学协同创新中心
Author(s):
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

关键词:
激光显微切割全自动毛细管Western blot蛋白质定量
Keywords:
laser microdissection automated capillary electrophoresis Western blotting protein quantification
分类号:
R329-33;R338.1;R446
文献标志码:
A
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。

Abstract:

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.

参考文献/References:

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更新日期/Last Update: 2018-10-31