胎鼠脑组织生长锥的提取及鉴定  

Extraction and identification of growth cone from brain tissue of fetal rats

作　　者：高媛[1,2] 纪志盛[3,2] 龚晓兵[4] 林宏生[3] 郭国庆[2] 

机构地区：[1]暨南大学附属第一医院消化科,广东广州510630 [2]暨南大学医学院 解剖学教研室,广东广州510630 [3]暨南大学 附属第一医院骨科,广东广州510630 [4]暨南大学 附属第一医院消化科,广东广州510630

出　　处：《暨南大学学报（自然科学与医学版）》2013年第4期378-384,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金项目(31170941)

摘　　要：目的：建立脑组织生长锥提取的方法。方法：采用梯度蔗糖离心的方法提取生长锥，透射电镜和扫描电镜观察分离物的形态，免疫荧光和免疫印迹检测GAP．43的表达。结果：梯度浓度蔗糖离心把脑组织悬液分离为3层，上层密度小，浓度位于上清至0．75mol／L蔗糖之间，中层密度较大，浓度位于0．75～1．00mol／L蔗糖之间，下层密度最大，浓度位于1．00—2．66mol／L蔗糖之间；透射电镜见上层分离物为大量着色浅的空泡状结构，颗粒大小较均匀，中、下层分离物为大量着色深的致密颗粒及不规则形态的细胞碎片，颗粒大小不均匀；扫描电镜见分离物颗粒均为白色、大小不一、形状不规则的颗粒，上层较中、下层颗粒体积大；免疫荧光及免疫印迹证实分离物中均有大量GAP－43表达，上层分离物几乎无GFAP表达，中、下层有少量GFAP表达。结论：梯度蔗糖离心的方法可以依据颗粒的大小分离脑组织，分离物最上层内的组分为生长锥。Aim ：To establish a method to extract growth cone from brain tissue. Methods： Using gra- dient sucrose centrifugation to extract growth cone, transmission electron microscopy and scanning electron microscopy to observe the morphology of the isolate, immunofluorescence and immunoblotting to detect the express of GAP-43 protein. Results： Gradient sucrose centrifugation isolated the brain tissue into three layers, the upper layer with low density, located between the supernatant and 0. 75 sucrose, the middle layer with higher density sucrose, located between 0. 75 to 1.0 mmol/L, the lower layer with the highest density sucrose, located between 1.00 to 2. 66 mmol/L. Observed by transmission electron mi- croscopy, the results showed that the upper layer contained a number of shallow-coloured vacuoles, and the particle size was uniform. The middle and lower layer contained a number of deep-coloured dense particles and irregular shaped cell debris, and the particle size was not uniform. The scanning electron microscopy images showed that the isolate were white, different sized, irregular shaped particles. The particles in the upper layer were larger than these in the middle and low layer. The immunofluorescence and immunoblotting results confirmed that all the isolate have a high expression of GAP-43. The particles in the upper layer almost has no GFAP expression, however, in the middle and lower layer, there were more GFAP expression. Conclusion：The gradient sucrose centrifugation can isolate the brain tissue according to the size of particles, and the components of the top isolate are growth cones.

关 键 词：梯度蔗糖离心 大鼠 生长锥 电镜 免疫荧光 免疫印迹 

分 类 号：Q189[生物学—神经生物学] Q954.67[生物学—普通生物学]