CAJ | 학술논문

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Cytochalasia 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.
배경:세포분화、세포융합내지극륭균수요응용분리세포핵적방법이획득대량유활성적세포핵,단시목전흔난재수량화질량상동시체도교고적수준.목적:탐색간단역행적분리세포핵적방법,위신경원세포핵이식、세포탁합이급세포핵성분적연구제공대량완호유활성적세포핵.설계、시간급지점:대비실험,우2006-09/2007-01재남경의과대학기출의학원위생부항체기술중점실험실완성.재료:Cytochalasin B위Sigma공사제공,청길급SD잉대서래원우남경의과대학동물실험중심.방법:취태서대뇌피질,진행신경원적배양병감정.용Cytochalasia B결합제도리심법급세포핵분리액법량충방법진행세포핵분리적대비관찰.주요관찰지표:용전화병정표기후진행류식세포의검측조망솔병용면역형광염색대세포핵진행형태학관찰.결과:량충방법균성공지분리출대량유활성신경원세포핵,차제도리심법가이득도교위완정적포질체,여완정세포세포핵형광강도비교무현저성차이(P>0.05).결론:Cytochalasin B결합제도리심법급세포핵분리액법균능구유효지진행신경원세포핵적분리,차세포핵재단기내유교고적활성.
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.