人髌下脂肪垫来源脂肪间充质干细胞的分离、培养及鉴定  

Human adipose-derived mesenchymal stem cells from the infrapatellar fat pad: isolation, culture and identification

作　　者：刘玉平[1] 刘涛[1] 王明明[1] 李明[1] 俞光荣[2] 

机构地区：[1]滕州市中心人民医院创伤骨科,山东省滕州市277500 [2]同济大学附属同济医院足踝外科,上海市200065

出　　处：《中国组织工程研究》2015年第41期6566-6571,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:髌下脂肪垫在膝关节手术中经常要部分切除,其可以作为脂肪间充质干细胞的重要来源。目的:探讨自髌下脂肪垫中分离、培养脂肪间充质干细胞的策略及细胞分子表面标记情况。方法:髌下脂肪垫组织取自膝关节镜手术的患者,以Ⅰ型胶原酶消化消化脂肪组织获取干细胞,用10%低糖DMEM培养基培养,利用MTT法测定不同代细胞增殖情况并绘制生长曲线。检测第5代细胞表面CD29及CD44的表达。结果与结论:培养24 h后可见原代细胞贴壁,1周后细胞呈纺锤型并且增殖速度加快,传代后的细胞贴壁及增殖细胞速度加快。生长曲线示第2及第5代的细胞增殖能力明显较第8代能力强。所取细胞能够分化为骨细胞和脂肪细胞。流式细胞仪检测结果显示第5代脂肪间充质干细胞重96.8%表达CD29,97.6%表达CD44。提示自髌下脂肪垫分离及提取脂肪干细胞简单易行,所得细胞的纯度及增殖能力均符合组织工程种子细胞的基本条件。BACKGROUND： Infrapatellar fat pad is often partially resected in the knee surgery, which can be used as an important source of adipose-derived mesenchymal stem cells. OBJECTIVE： To explore the strategies of isolation, culture, and identification of adipose-derived mesenchymal stem cells from the infrapatellar fat pad and to detect the expression of cell surface markers of human adipose-derived stem cells. METHODS： Infrapatellar fat pad was obtained from patients undergoing knee arthroscopy surgery, and attached cells were obtained from adipose tissue by using collagenase I. Cells were cultured in 10% low-sugar DMEM. Stem cells proliferation was detected by means of MTT and then, cell growth curve was made. The obtained cells were induced and differentiated into adipocytes and osteocytes. Expressions of cell surface markers CD29 and CD44 were detected. RESULTS AND CONCLUSION： A few of attached cells were observed after cultured 24 hours. Cells proliferated faster and exhibited spindle shape after 1 week. Cell adherence and proliferation were speeded up after subculture. Growth curve of cells exhibited that the passages 5 and 2 cells had higher reproductive activity than passage 8 cells. The obtained cells can be induced and differentiated into adipocytes and osteocytes. Results from flow cytometry showed that 96.8% passage 5 cells expressed CD29 and 97.6% expressed CD44. These findings indicate that high-purity adipose-derived mesenchymal stem cells with high reproductive ability are easy to be isolated from the infrapatellar fat pad, which may be a kind of ideal seed cells for cartilage tissue engineering.

关 键 词：干细胞 间质干细胞 细胞培养技术 抗原 表面 组织工程 脂肪干细胞 脂肪间充质干细胞 细胞培养 种子细胞 表面抗原 

分 类 号：R394.2[医药卫生—医学遗传学]