胎鼠皮质神经元细胞核的制备和鉴定  

Separation and identification of the karyoplasts of cortical neurons of embryonic rats

作　　者：周竹娟[1] 郑健[1] 徐炎[1] 

机构地区：[1]第三军医大学新桥医院神经内科,重庆400037

出　　处：《卒中与神经疾病》2008年第2期98-102,共5页Stroke and Nervous Diseases

基　　金：国家自然科学基金资助项目(30270490);重庆市科技计划项目(7452)

摘　　要：目的 建立制备皮质神经元细胞核的方法，以便用于核移植研究。方法 （1）采用胎龄17d的胎鼠进行皮质神经元的分离培养，用抗MAP2和NeuN免疫细胞化学法进行鉴定；（2）用梯度离心法制备神经元细胞核，并用HE染色、免疫荧光染色、透射电镜检查等方法进行鉴定，用台盼蓝染色检测其活性。结果 （1）胎鼠皮质神经元呈多角形，伸出多个突起，MAP2染色显示神经元细胞浆和突起呈免疫反应阳性，NeuN染色显示神经元细胞核呈免疫反应阳性，部分核周胞质和部分近端突起也呈免疫反应阳性，MAP2和NeuN阳性细胞比例均在90％左右；（2）HE染色和Hoechst33342免疫荧光染色显示神经元梯度离心后在22％～30％Ficoll400梯度层可见到大量圆点状细胞核，台盼蓝染色显示活细胞核比例为99．6％；在22％～30％Ficoll400梯度层细胞透射电镜标本中可观察到圆形的细胞核，在细胞核的周围仅见到一层薄薄的胞浆成分。结论 梯度离心法是制备皮质神经元细胞核的可靠方法。Objective To establish a method of separating karyoplasts of cortical neurons by centrifugation through a Ficoll density gradient for nuclear transfer. Methods （1） The neurons from the cerebral cortex of embryonic 17d rats was isolated and identificated by immunostaining with MAP2 and NeuN antobodies. （2） Separation of karyoplasts of cortical neurons was completed by centrifugation through a Ficoll density gradient. The karyoplasts were identified by HE staining, immunofluorescence staining and transmission electron microscopy. The cell viability of karyoplasts was estimated by trypan blue exclusion test. Results （1）The cortex neurons of embryonic rat had polygonal morphology with several processes. MAP2 immunoreactivity was observed in cytoplasm and processes of neurons. NeuN immunoreactivity was observed in nucleus, some cytoplasm around the nucleus and proximate processes. The ratios of MAP2-postive and NeuN-postive neurons were about 90% . （4）Karyoplasts were collected from the 22% ,25% and 30% regions after centrifuging MSCs through a Ficoll gradient by HE staining and immunofluorescence labeling with Hoechst 33342. The ratio of viable karyoplasts estimated by trypan blue exclusion test was 99. 6%. The round karyoplast surrounded by a very thin layer of cytoplasm enclosed within a plasma membrane was observed in 22-30% Ficoll gradient sam- ple with transmission electron microscope. Conclusions Centrifugation through a Ficoll density gradient is a reliable method to separate the karyoplasts of cortical neurons.

关 键 词：神经元 细胞核 核移植 

分 类 号：Q813[生物学—生物工程] R741[医药卫生—神经病学与精神病学]