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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:CytochalasinB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:CytochalasinB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。
Background: Cell differentiation, cell fusion and even cloning all require the use of a method of separating the nuclei to obtain a large number of active nuclei. However, it is currently difficult to achieve a higher level of both quantity and quality. OBJECTIVE: To explore a simple and easy method of separating nuclei and provide a large number of intact and active nuclei for the study of neuronal cell nuclear transfer, cell disassembly and nuclear component. DESIGN, TIME AND SETTING: The comparative experiment was performed at the Key Laboratory of Antibody Technology, Ministry of Health, School of Basic Medical Sciences, Nanjing Medical University from September 2006 to January 2007. MATERIALS: CytochalasinB was provided by Sigma Company. The clean-grade SD pregnant rats were obtained from Animal Experimental Center of Nanjing Medical University. Methods: Fetal cortex was taken and neurons were cultured and identified. Cytochalasin B combined with gradient centrifugation and nuclear separation liquid two methods of nuclear isolation comparative observation. MAIN OUTCOME MEASURES: After being labeled with propidium iodide, the apoptosis rate was detected by flow cytometry and the morphology of the nucleus was observed by immunofluorescence staining. Results: A large number of nuclei of active neurons were successfully separated by both methods. The intact cytoplasm could be obtained by gradient centrifugation. There was no significant difference between the two methods (P> 0.05). CONCLUSION: Cytochalasin B combined with gradient centrifugation and nuclei separation liquid method can effectively separate the nucleus of neurons, and the nucleus has high activity in a short period of time.