大量活性神经元细胞核的分离技术

背景：细胞分化、细胞融合乃至克隆均需要应用分离细胞核的方法以获得大量有活性的细胞核，但是目前很难在数量和质量上同时达到较高的水准。目的：探索简单易行的分离细胞核的方法，为神经元细胞核移植、细胞拆合以及细胞核成分的研究提供大量完好有活性的细胞核。设计、时间及地点：对比实验，于2006-09／2007-01在南京医科大学基础医学院卫生部抗体技术重点实验室完成。材料：Cytochalasin B为Sigma公司提供，清洁级SD孕大鼠来源于南京医科大学动物实验中心。方法：取胎鼠大脑皮质，进行神经元的培养并鉴定。用Cytochalasin B结合梯度离心法及细胞核分离液法两种方法进行细胞核分离的对比观察。主要观察指标：用碘化丙啶标记后进行流式细胞仪检测凋亡率并用免疫荧光染色对细胞核进行形态学观察。结果：两种方法均成功地分离出大量有活性神经元细胞核，且梯度离心法可以得到较为完整的胞质体，与完整细胞细胞核荧光强度比较无显著性差异（P〉0．05）。结论：Cytochalasin B结合梯度离心法及细胞核分离液法均能够有效地进行神经元细胞核的分离，且细胞核在短期内有较高的活性。

Isolation technique for harvesting a great amount of active neuronal nuclei

BACKGROUND: A method for isolation of cell nuclei is needed to collect a great many of active nuclei during cell differentiation, cell confluence and clone, but it is difficult to reach a higher level in quantity and quality.OBJECTIVE: To investigate a simple easy method for isolation of cell nuclei, and to provide a great quantity of active cell nuclei for studying neuronal nucleus transplantation, cell isolation-fusion and nucleus composition.DESIGN, TIME AND SETTING: The controlled experiment was performed at the Key Laboratory of Antibody Technique, Health Ministry of China, College of Preclinical Medicine, Nanjing Medical University from September 2006 to January 2007.MATERIALS: Cytochalasin B was purchased from Sigma, USA. Pregnant Sprague Dawley (SD) rats of clean grade were obtained from the Animal Experimental Center of Nanjing Medical University.METHODS: Neurons were collected from cerebral cortexes of fetal rats, cultured and identified. Cell nuclei were isolated by gradient centrifugatiou combined with Cytochalasin B treatment and cell nucleus separating medium, and then compared.MAIN OUTCOME MEASURES: After labeling with propidium iodide, apoptotic rate was measured by flow cytometry; Cell nuclei were observed by immunofluorescence staining.RESULTS: A considerable number of active neuronal nuclei were successfully harvested. Intact cytoplasts were collected by gradient centrifugation, and no significant difference in fluorescence intensity was detected compared with intact nuclei (P > 0.05).CONCLUSION: Neuronal nuclei are effectively isolated by gradient centrifugation and separating medium combined with Cytochalasin B. Cell nuclei have a high activity in a short term.