【摘要】 背景:干细胞是具有自我更新能力和高度增殖能力和多种分化潜能的较原始细胞,在一定的条件下,干细胞可以分化成机体内的多种功能细胞。胰岛干细胞和脐血间充质干细胞可以治疗相应系统的疾病,但是目前有关这两类干细胞的培养仍有困难。目的:探讨胰岛干细胞和脐血间充质干细胞分离和培养的方法,并观察在培养过程中两类干细胞的形态学变化。设计:完全随机分组设计,重复测量实验。单位:郑州大学医学院生理学教研室干细胞研究中心材料:实验于2004-04/2005-01在郑州大学医学院干细胞中心完成。选用鼠龄1～3d新生SD大鼠10～15只进行胰岛干细胞培养,采集年龄24～35岁的足月妊娠健康产妇(经过产妇知情同意)新鲜脐带血进行脐血间充质细胞的培养。方法:取新生SD大鼠,无菌条件下剖腹取胰,眼科剪反复剪切后V型胶原酶消化分离胰岛。连续转瓶法纯化胰岛。取新鲜采集脐带血,用1.077g/cm3的淋巴细胞分层液,密度梯度离心法分离脐血单个核细胞。将胰岛细胞和脐血单个核细胞接种于37℃,体积分数0.05CO2培养箱内培养。于不同时间观察细胞形态的变化并通过流式细胞仪检测细胞表面分子的表达情况。主要观察指标:胰岛干细胞和脐血间充质干细胞的分离培养方法以及在细胞生长过程中的形态学变化。结果:①培养中可见梭形胰岛祖细胞贴壁呈极性生长,并且不断增殖,约12～14d长满瓶底,可连续多次传代。撤去生长因子和血清后有圆形细胞团开始形成。②从脐血中分离出的单个核细胞,培养中先出现大量的造血细胞集落,瑞士染色显示这些细胞大多数为粒系的集落,7d后出现贴壁的扁平状的上皮样细胞和长梭形的成纤维样细胞,同时有大量的破骨样细胞混杂。随着培养时间的延长,细胞数目不断增加。结论:胰岛干细胞和脐血间充质干细胞可以在体外分离和培养,用于进一步相关实验工作。更多还原

【Abstract】 BACKGROUND Stem cells are relatively primitive cells possessing the capabilities of self-renewal high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells MSCs may serve therapeutic purpose clinically but they are still difficult to culture in vitro at present. OBJECTIVE To explore the method for isolation purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro. DESIGN Completely randomized experiment with repeated measurement. SETTING Stem Cell Research Center Teaching and Research Division of Physiology Medical School of Zhengzhou University.MATERIALS This experiment was conducted in the Stem Cell Research Center Teaching and Research Division of Physiology Medical College of Zhengzhou University between April 2004 and January 2005. Ten to fifteen newborn SD rats 1-3 days were selected for culture in vitro of pancreatic stem cells and fresh umbilical cord blood was collected from healthy woman 24-35 years old with informed consent at full-term delivery for culture in vitro of umbilical blood SMCs. METHODS The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium with density of 1.077 g/cm3. The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs and their morphological changes during culture in vitro. RESULTS During culture in vitro the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones most of which proved to be granulocyte clones by Switzerland staining. Seven days later flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged the cell number increased steadily. CONCLUSION Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.更多还原