骨髓间质干细胞的胞质体制备及其活性

背景：细胞质在细胞分化过程中起着决定作用，但目前的实验很难获得大量的、 有活性的胞质体。 目的：分析影响大鼠骨髓间质干细胞胞质体制备的因素，并观察胞质体活性，以 期为细胞重组提供实验依据。 设计、时间及地点：以细胞为观察对象的多因素设计实验，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：选用1月龄SD大鼠，用于分离骨髓间充质干细胞。 方法：骨髓间质干细胞经分离培养、扩增后，用松胞素B、秋水仙碱结合离心术制 备胞质体。 主要观察指标：应用苏木精-伊红、Alexa-Flour?488anti-rat CD106及Bis- Benzimide染色进行形态学观察，计算胞质体纯度；应用Rhodamine123标记后用流 式细胞仪检测胞质体中线粒体膜势能。 结果：①胞质体纯度可达95%。80%的胞质体在一两个小时内恢复原来形态，但其 生活力不会超过18 h。胞质体活性随时间延长而下降。②骨髓间质干细胞的胞质 体制备与松弛素B、秋水仙碱剂量、离心速度、离心时间，特别是离心温度有关。 结论：骨髓间质干细胞的胞质体制备可受多因素影响。当胞质体具有活性，具有 有核细胞的一些行为特征时，可以进行细胞重组。

Preparation and activity of cytoplasts from bone marrow mesenchymal stem cells

BACKGROUND: The cytoplasm plays a decisive role in the process of cell differentiation. However, it is difficult to obtain a large number of live cytoplasm in present study. OBJECTIVE: To analyze relevant factors of preparation of cytoplasts from rat bone marrow mesenchymal stem cells, and to observe activity of cytoplasts to provide experimental evidences for cell recombination. DESIGN, TIME AND SETTING: The multiple factor design cell experiment was performed at the Key Laboratory of Antibody Technique, Ministry of Health, Basic Medical College, Nanjing Medical University from September 2006 to January 2007. MATERIALS: One-month-old Sprague Dawley rats were used for isolating bone marrow mesenchymal stem cells. METHODS: After bone marrow mesenchymal stem cells were isolated, cultured and amplified, cytoplasts were prepared by cytochalasin B and colchicine treatment followed by centrifugation. MAIN OUTCOME MEASURES: Morphology was observed using hematoxylin-eosin staining, Alexa- Flour?488anti-rat CD106 and Bis-Benzimide staining to calculate cytoplast purity. Mitochondrial membrane potential was detected by flow cytometry following labeling with Rhodamine123. RESULTS: The purity of cytoplasts was up to 95%. Eighty percent of cytoplasts recovered the original shape between one and two hours. However, its lifetime would not be more than 18 hours. The activity of cytoplasts would decrease over time. The preparation of cytoplasts from bone marrow mesencymal stem cells is related to dose of cytochalasin B, dose of colchicines, speed and time of centrifugation, especially, temperature of centrifuge. CONCLUSION: Preparation of cytoplasts from bone marrow mesenchymal stem cells is affected by multiple factors. Cytoplasts have activity and behavior characteristics of nucleated cells. Cytoplasts can do cell reorganization.