小鼠骨髓间充质干细胞的分离与纯化培养的实验研究  

Cultivation,retrieval and purification of mouse MSCs

作　　者：仝识非[1] 宋治远[1] 姚青[1] 万瑛[2] 邹丽云[2] 

机构地区：[1]第三军医大学西南医院心血管内科 [2]第三军医大学基础医学部全军免疫学研究所,重庆400038

出　　处：《第三军医大学学报》2007年第10期907-910,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30370585)~~

摘　　要：目的探索小鼠骨髓间充质干细胞的体外纯化培养方法。方法先采用直接贴壁法培养的小鼠股、胫骨髓细胞,再用免疫磁珠法分选第3代中的CD11b-细胞。取分选纯化细胞进行形态学观察,并检测细胞在不同诱导条件下向成骨细胞及脂肪细胞的分化能力。结果①原代及传代培养的小鼠骨髓贴壁细胞多呈梭形,部分呈不规则型,其中含有较多的CD11b+细胞;磁珠分离后的纯化细胞呈现纺锤型、星型及不规则型等多种形态,细胞质丰富,核仁明显,细胞平行排列或漩涡状生长;②成骨诱导3周后mMSCs分化为成骨细胞,茜素红S染色有橘红色磷酸盐胞外基质沉积;③随着成脂化诱导时间的延长,细胞逐渐增大,油红O染色可见胞质内大量橙红色脂肪空泡。结论单纯的贴壁及传代培养不能纯化小鼠骨髓间充质干细胞,贴壁与免疫磁珠分离相结合才是一种有效的mMSCs体外分离和纯化方法。Objective To develop new methods to cultivate, retrieve and purify mouse mesenchymal stem cells （mMSCs）. Methods Bone marrow was collected from 2-month-old Kunming mice by flushing femurs and tibias with complete medium of DMEM-LG. Cells were plated in a Petri dish. After 24 hours, non-adherent cells were removed by two to three washes with PBS, adherent cells were further cultured in complete medium and retrieved by trypsinisation with 0. 25% trypsin for 5 min at 37℃. The treated adherent cells were cultivated with 3 × dilution for further generations. CD11b-negative cells were retrieved from the collected adherent cells of 3 ^rd generation by using immunomagnetic microbeads, and continued to be cultured in complete medium. After the cultured cells were retrieved, their morphology and their ability of osteoblastic differentiation and adipocytic differentiation were examined. Results Most of mMSCs from 1^st generation were of shuttle shape, some of irregular shape. After treatment with magnetic microbeads and several generations, mMSCs were of spindle, star and irregular shape. These cells were of rich cytoplasma, clear nucleolus, and grew in parallel or vortex. The cultured adherent cells from the first and subsequent generations had plenty of CD11 b-positive bloocling-making cells. After 20-day osteoblastic induction, mMSCs differentiated into bone cells, which showed orange phosphate in extracellular matrix by Alizarin red S staining, mMSCs could differentiate into lipocytes. The size of cells increased along with fat-developing induction period. These cells showed many orange fatty follicles with O Red Oil dyeing. Conclusion Pure mMSCs can not be retrieved by either adhering method or generation cultivation method separately. The combined methods of adhering, immunomagnetic microbeads, and serial subcuhivation is effective in vitro in retrieve mMSCs.

关 键 词：间充质干细胞 小鼠 免疫磁珠 骨髓 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]