培养大鼠海马神经元树突发育的活细胞成像和量化分析

Live imaging and quantitative analysis of dendritic development of cultured rat hippocampal neurons

目的：采用活细胞成像法测量培养海马神经元的树突生长、分支复杂程度和树突丝的运动性，以及定量分析树突树发育的形态学特征。方法：出生后第1天进行海马神经元培养，于第5天转染绿荧光蛋白（GFP）标记的纤维型肌动蛋白（GFP—F—Actin）和定位在膜上的GFP（F—GFP），观察培养第7～14天树突的发育情况并进行量化分析。结果：培养第7～9天可以观察到高密度的树突丝活跃的运动，树突丝的平均密度分别为（10．78±3．78）个／100μm、（10．68±2．96）个／100μm、（9．99±3．67）个／100μm（P〉0．05），有（30．18±14．03）％到（87．36±20．88）％的树突丝运动活跃（P〈0．001）；第7～14天树突树的总长度从（410．74±185．98）μm增长为（1238．21±418．32）μm（P〈0．001），树突树的总分支数从（18．93±7．23）支增加为（33．60±10．46）支（P〈0．001）；培养第14天树突棘的密度为（37．17±6．46）个／100μm。结论：结合荧光蛋白转染和活细胞成像的方法，可动态、连续地观察，并定量分析发育过程中树突丝、树突树和树突棘的形态学特征，是体外观察神经元发育的理想方法。

Objective： To measure mobility of dendritic filopodia, complexity of dendritic arborization using method of live imaging in cultured rat hippocampal neurons and to analyze their morphological characters quantitatively. Methods： Vectors expressing Green Fluorescent Protein- Fibrous Actin （GFP-F-Actin） and F-GFP were co-transfected into cultured rat hippocampal neurons at 5 d in vitro （DIV 5）. Neurons expressing GFP were photographed and analyzed with Metamorph software. Results. Dendritic filopodia was observed to move actively from DIV 7 to DIV 9. The mean density of filopodia was （10.78±3.78）/100 μm, （10. 68±2.96）/100μm and （9.99 ±3.67）/100 μm （P〉 0.05）, and there were （30.18 ± 14.03） % to （87.36 ± 20.88）% filopodia were mobile （P〈0. 001）. During DIV 7-DIV 14,the total length of dendritic branches grew from （410.74±185.98）μm to （1238.21±418.32）μm （P〈0. 001） and the number of dendritic branches increased from 18.93± 7.23 to 33.60± 10.46 （P〈0. 001）. The density of spine was （37.17±6.46）/100 μm at DIV 14. Conclusion： The combination of live imaging with quantitative analysis is a useful method to study dendritic morphological development in vitro,including indicators of dendritic filopodia,dendritic arborization and spines.