脐带间充质干细胞分离培养方法的优化.docVIP

  脐带间充质干细胞分离培养方法的优化# 余永春1,2，王伟新1,3，孙洁1,4，敬伟1，刘磊1** （1. 四川大学口腔疾病研究国家重点实验室，成都 610041； 5 10 15 20 25 30 35 40 45 2. 广州医学院附属第一医院口腔科，广州 510120； 3. 新乡医学院第一附属医院口腔科，河南 新乡 453000； 4. 广东省口腔医院正畸科，广州 510280） 摘要：目的: 探讨脐带间充质干细胞分离培养的最佳方法。方法: I 型胶原酶加胰酶、II 型胶 原酶加胰酶、II 型胶原酶透明胶原酶加胰酶和贴壁法分离培养人脐带间充质干细胞，并用含 10%胎牛血清的 DMEM/F12 进行培养。研究中调整血清浓度 15%或者 20%；调整培养基为 低糖 DMEM 或者高糖 DMEM；添加表皮生长因子和重组碱性成纤维生长因子。倒置显微镜 观察细胞形态。MTT 法绘制细胞生长曲线。结果: 仅 I 型胶原酶加胰酶方法可高效成功地分 离人脐带间充质干细胞；其他分离方法结果均为阴性，改变培养基、血清浓度和添加生长因 子都未见分离出细胞。结论：采用 I 型胶原酶加胰酶方联合消化法以及含 10%胎牛血清的 DMEM/F12 可以快速高效的分离培养脐带间充质干细胞。 关键词：脐带间充质干细胞；分离和培养方法；酶消化；贴壁法 中图分类号：R329 Optimized research on isolation and cultivation of umbilical cord mesenchymal stem cells YU Yongchun1,2, WANG Weixin1,3, SUN Jie1,4, JING Wei1, Liu Lei1 (1. State Key Laboratory of Oral Diseases, Sichuan University, ChengDu 610041; 2. Department of Stomatolgoy, First Affiliated Hospital of Guangzhou Medical College, GuangZhou 510120; 3. Department of Stomatolgoy, First Affiliated Hospital of Xinxiang Medical College, HeNan XinXiang 453000; 4. Department of Orthodontics, Guangdong Provincial Stomatological Hospital, GuangZhou 510280) Abstract: Objective: To investigate the best method to isolate and cultivate umbilical cord mesenchymal stem cells. Methods: Type I collagenase plus trypsin enzyme, type II collagenase plus trypsin, type II collagenase plus trypsin and hyaluronidase and adherent method were used to isolate human umbilical cord mesenchymal stem cells, and DMEM/F12 containing 10% fetal bovine serum (FBS) was used to cultivate it. During the research, we changed the culture conditions to enable the cells digested of the dissociated from umbilical cord to adhere and grow. The serum concentration was adjusted to 15% or 20%; the medium was adjusted to low glucose DMEM or high glucose DMEM medium plus epidermal growth factor and recombinant basic fibroblast growth factor. Inverted microscope was used to check the cell. MTT method was used to depict growth curve of the cells. Results: O