冻存复苏人脐带间充质干细胞体外扩增和向脂肪细胞分化的研究  

Differentiation of cryopreserved human Umbilical Cord Mesenchymal Stem Cells into adipogenic cells

作　　者：肖宏涛[1] 刘毅[1] 陈克明[2] 

机构地区：[1]兰州军区兰州总医院烧伤整形科 [2]兰州军区兰州总医院创伤外科研究所,甘肃兰州730050

出　　处：《中国美容医学》2009年第4期484-486,共3页Chinese Journal of Aesthetic Medicine

基　　金：国家自然科学基金(30872689);全军"十一五"医学科学技术研究面上项目(06MA079);甘肃省自然科学基金(3ZS061-A25-099)

摘　　要：目的:分离培养人脐带间充质干细胞(human Umbilical Cord Mesenchymal Stem Cells hUCMSCs),观察其冻存复苏后向脂肪细胞定向分化的能力。方法:将剔除动静脉的新鲜人脐带组织切成小块培养,得到贴壁细胞,观察细胞生长及检测其表面抗原。将第1代的hUCMSCs采用梯度冷冻技术冻存5个月,复苏后培养至第12代时,加入成脂诱导剂培养。当诱导至21天时行油红O染色,7天及14天时用RT-PCR技术分别检测成脂转化基因过氧化物酶增殖剂受体(PPARγ-2)和脂蛋白酯酶(LPL)。结果:组织块培养法收获的单个核细胞培养传代后,能获得均一贴壁的间充质干细胞,hUCMSCs冻存复苏后,活细胞约为86%,流式细胞仪分析这些细胞表达CD13、CD44和CD71等MSCs标志物,不表达CD14、CD34和HLA-DR表面抗原。复苏细胞在成脂诱导剂的作用下,细胞中有脂滴产生,通过油红O染色显示细胞核周围有脂滴聚集,通过RT-PCR检测有LPL及PPARγ2mRNA的表达。结论:采用组织块贴壁培养法分离获得的人脐带间充质干细胞可冷冻保存。复苏后培养至第12代仍具有向脂肪细胞分化的潜能,可作为脂肪组织工程种子细胞来源。Objective To isolate and culture the human Umbilical Cord Mesenchymal Stem Cells （hUCMSCs） and study the potential to differentiate into adipogenic cells after they were frozen and thawed. Methods After stripping off arteries and veins, the remaining parts of umbilical cord were cut into small sections and plated in 100mm culture capsule. Adherent cells coming out of fragments and the cell growth was observed and its surface antigen was detected. The one-passage hUCMSCs were preserved in liquid nitrogen by reducing the temperature gradually. Five months later, cells were thawed. The 12-passage hUCBMSCs were treated with differentiation medium ,induceed after 21 days by Oil Red-O analysis,RT-PCR analyes for peroxisome proliferator - activated receptor-γ2 and lipoproteinesterase induceed after 7 days and 14 days respectively. Results The adherent homogeneous hUCMSCs were obtained with the cultivation of small tissue sections and the living cells of hUCMSCs were86% after having been frozen and cells after thawing.FACS revealed that these cells expressed common markers of MSCs, such as CD13, CD44 and CD71, but did not express markers CD14,CD34and HLA-DR. Adipogenic differentiation was seen as Oil red O-positive cells and was also analyzed by RT-PCR, in which the adipocyte marker LPL and PPAR-γ2 was expressed in the adipogenic formula-treated cells. Conclusions hUCMSCs can be obtained by means of the cultivation of small tissue sections, and preserved by cryopreservation. The hUCMSCs frozen and thawed were cultured to 12-passage still having adipogenic differentiation potential,and that cryopreserved hUCMSCs can be used for adipose tissue engineering.

关 键 词：冻存 脐带 间充质干细胞 分化 脂肪细胞 

分 类 号：Q813.1[生物学—生物工程]