【作者】 吴微；

【导师】 孙长凯；

【作者基本信息】 大连医科大学 ， 生理学， 2011， 硕士

【摘要】 目的:关于脑保护和脑损伤修复研究中,一般采用在体动物实验和体外细胞培养两种。在体实验虽然能保证与人体内环境的高度相似性,但由于其环境复杂,不可控因素较多,无法进行对单一干扰因素的分析。而单一的细胞培养忽略了脑内不同细胞(脑毛细血管内皮细胞、星形胶质细胞及神经元)之间的相互作用,因此共培养技术应运而生。但大多采用细胞系或来自不同种属动物的原代培养细胞共培养,已有文献指出这两种方式存在着许多不可避免的差异,这些差异往往会影响实验结果。为寻求一种更加客观有效的实验平台,本实验运用Transwell技术建立SD(Sprague-Dawley)大鼠的大脑毛细血管内皮细胞(Endothelial cell,E)、星形胶质细胞(Astrocyte,A)及神经元(Neuron, N)三细胞原代共培养模型系统。与以往实验相比,原代培养的细胞仍保留原有在体部分特性,可以实现模拟类似于血脑屏障结构及其客观微环境下的存活、生长、分化、功能、损伤、修复及相互作用。为将来脑保护与脑损伤修复研究提供了一个科学可信的新平台。方法:通过本实验室的经验积累以及参考大量文献的实验方法,作者对SD大鼠的大脑毛细血管内皮细胞、星形胶质细胞、神经元进行原代培养,用葡萄糖转运体1(Glucose Transport 1,GLUT-1)胶质原纤维酸性蛋白(Glial Fibrillary Acidic Protein ,GFAP)、微管相关蛋白2(Microtubule-Associated Protein 2,MAP-2)分别鉴定标记大脑毛细血管内皮细胞、星形胶质细胞及神经元,并计算其纯度。利用Transwell将三种细胞按一定时间和空间的顺序建立EAN三细胞共培养模型系统。当系统稳定后,利用激光共聚焦成像系统,分别拍摄单独培养和EAN系统中星形胶质细胞的GFAP免疫荧光图像直观描绘其形态学特征,利用图像分析软件Image-pro Plus6.0对其GFAP阳性结构中心聚集面积和投射周围的长度及面积进行测量,根据统计学方法客观分析其形态学特征,同时利用三维重建技术,分析星形胶质细胞GFAP阳性结构投射在Transwell半透膜微孔中的生长情况。结果:一、EAN模型中星形胶质细胞的GFAP阳性结构中心聚集面积为940.00±178.15μm2。单独培养的星形胶质细胞的GFAP阳性结构中心聚集面积为1946±173.01μm2。两者差异具有统计学意义(P<0.05)。二、EAN模型中星形胶质细胞的GFAP阳性结构投射周围长度为156.33±17.64μm,面积为2196.00±241.50μm2。单独培养的星形胶质细胞的GFAP阳性结构投射周围长度为108.48±13.33μm,面积为515.50±156.84μm2。两者GFAP阳性结构投射周围长度差异具有统计学意义(P<0.05),两者GFAP阳性结构投射周围面积差异具有统计学意义(P<0.05)。三、在Transwell中半透膜的微孔发现有星形胶质细胞的GFAP阳性结构存在。结论:一、在EAN三细胞共培养的模型系统中,星形胶质细胞GFAP阳性结构形态与单独培养条件下存在差异。与之比较,EAN模型中星形胶质细胞的GFAP阳性结构中心聚集面积减小,而GFAP阳性结构投射周围长度和面积均增大。二、EAN模型中星形胶质细胞GFAP阳性结构能够沿着Transwell中半透膜的微孔,向种植有大脑毛细血管内皮细胞方向生长。更多还原

【Abstract】 Objective:During the research of the neuroprotection and the neuroregeneration, that often uses two different methods, which are in vitro experiment and in vivo experiment. The cell still retained similar original body characteristics in vivo experiment, but, its environment is complicated, which cannot control a several of factors, and it also cause the barrier of analysis to the interference of single factor. However, monoculture has neglected the interaction of different cells in the brain (such as, brain capillary endothelial cells, astrocytes and neurons). As a result, the co-culture technique has appeared. Generally, the primary culture cells introduce from the different species of animal to using the co-culture. According to several scientific references, those two methods have ineluctable discrepancy that usually affects an experiment result. In order to gain more objective and effective experiment platform, the Transwell technique of this experiment usage creates a three kind of the Sprague-Dawley (SD) rat’s primary culture cells (brain capillary endothelial cells, astrocytes and neurons--EAN) in the co–culture model system. From different previous studies, the primary culture cell still retained original partial characteristics in vivo and it also realizes the simulation to be similar under the blood brain barrier structure and the objective micro-environment survival, the growth, the differentiation, the function, the damage, the repair and the interaction. Therefore, it can enhance experiment’s accuracy and the reliability, has provided a new scientific credible platform for the future neuroprotection and neuroregeneration research.Methods:According to several scientific references and the laboratory work experience, the author chooses the SD rat brain capillary endothelial cells, astrocytes and neurons that can be primary cultured, through the GLUT-1, GFAP and MAP-2 to mark brain capillary endothelial cell, astrocyte and neuron separately to measure the purity of cells. These cells are planted at a regular time and space via the transwell in sequence to establish EAN three cell co–culture model system. When the system became stable, using the confocal microscopy system, photographs astrocyte’s GFAP immunity fluorescence ICON to intuitively describe its change of morphological character in the monoculture and the EAN model system separately. By using Image-pro Plus6.0 software, it can measure the peripheral projection length and area of astrocyte’s GFAP positive structure and the central complex area of astrocyte’s GFAP positive structure. Basic on the statistics method, analysis its change of morphological character objectively. Simultaneously using the three dimensional reconstruction technology, analysis the astrocyte’s GFAP positive structure that projects on the Transwell semi-permeable membrane pore growth status.Results:1. The central complex area of astrocyte’s GFAP positive structure is 940.00±178.15μm2 in the EAN model, while in monoculture is 1946±173.01μm2. Both difference has statistics significance (P<0.05).2. The peripheral projection length of astrocyte’s GFAP positive structure is 156.33±17.64μm, and the area is 2196.00±241.50μm2 in the EAN model. The peripheral projection length of astrocyte’s GFAP positive structure is 108.48±13.33μm, and the area is 515.50±156.84μm2 in monoculture. Both the peripheral projection length of GFAP positive structure difference has statistics significance (P<0.05), and also both the peripheral projection area of GFAP positive structure difference has statistics significance (P<0.05).3. During Transwell the semi-permeable membrane pore that discover the existence of the astrocyte’s GFAP positive structure.Conclusions:1. Using the primary culture the brain capillary endothelial cell, the astrocyte and the neuron established in the EAN three cell co–culture model system, which the GFAP positive structure shape of astrocyte compare with the condition of the sole culture existing difference. Compare to monoculture of astrocyte, the astrocyte’s GFAP positive structure center of complex area reduces in the EAN model, but the periphery of the GFAP positive structure projection the length and the area increase.2. In the EAN model the astrocyte’s GFAP positive structure can grow along Transwell in the semi-permeable membrane pore,which cultivates the direction of the brain capillary endothelial cell to growth.更多还原