人脂肪间充质干细胞生物学特征及体外向类角质细胞的分化  

Biological characteristics of human adipose tissue-derived mesenchymal stem cells and the differentiation into keratinocyte-like cells in vitro

作　　者：王先成[1] 乔群[2] 管利东[3] 王韫芳[3] 裴雪涛[3] 何利娟[3] 

机构地区：[1]中南大学湘雅二医院烧伤整形外科,湖南省长沙市410011 [2]中国医学科学院北京协和医院整形外科,北京市100730 [3]解放军军事医学科学院输血研究所干细胞研究中心,北京市100850

出　　处：《中国组织工程研究与临床康复》2007年第37期7333-7336,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家重点基础研究发展规划项目(2001CB509906和2005CB522702);国家高技术计划领域重大专项(2002AA205051和2003AA205160)资助项目;北京市科委课题(H020220010190)~~

摘　　要：目的:在体外不同培养条件下,脂肪来源间充质干细胞可以向成骨细胞、成软骨细胞、神经细胞、内皮细胞、肌肉细胞、脂肪细胞和肝脏细胞等不同胚层来源的细胞分化。观察脂肪间充质干细胞在体外培养并向类角质细胞分化的能力。方法:实验于2005-06/2006-03在解放军军事医学科学院完成。实验方法:取20～45岁健康女性(已取得患者同意)脂肪组织行脂肪抽吸术后的脂肪颗粒。参照Zuk等方法分离脂肪间充质干细胞。取培养的第3代细胞,采用流式细胞仪鉴定细胞表面分子及细胞周期。取状态良好、融合至瓶底80%的第20代细胞,在5mg/L的秋水仙素处理后采集分裂细胞,以Giemsa染色进行核型分析。取第3代细胞以5μm维甲酸和5μg/L表皮细胞生长因子培养基向角质细胞诱导分化。对照组用含体积分数为0.05胎牛血清的L-DNMEM培养基。实验评估:采用倒置显微镜观察细胞形态,免疫组织化学方法检测角质细胞的标志物CK19。结果:①脂肪来源间充质干细胞表面分子CD29、CD90、CD44呈阳性,而CD34呈阴性。②细胞增殖曲线呈S型,接种的前3d细胞处于滞留期,3d后进入对数生长期,6d后进入平台期,细胞的群体倍增时间约为29h。处于对数生长期的细胞80%以上处于G0和G1期。③细胞染色体核型分析显示,染色体数目2n=46,核型46,xx,为女性正常核型。④对照组细胞贴壁后,呈成纤维细胞样,细胞呈长梭形,排列成漩涡状,贴壁较好。诱导组从诱导的第3天开始,细胞逐渐变成不规则,诱导第10天出现铺路石改变,形似角质细胞。⑤免疫组织化学方法检测显示,部分细胞专一地对CK19抗体呈阳性反应,有棕色染色。结论:可从人脂肪抽吸物中分离出间充质干细胞,脂肪间充质干细胞在体外一定条件下可向类角质细胞分化。AIM： Adipose tissue-derived mesenchymal stem cells （ADMSCs） can differentiate into osteoblasts, chondroblast, neural cells, endothelial cells, muscle cells, adipose cells and hepatic cells under certain conditions in vitro. This article is designed to investigate the ability of ADMSCs cultured in vitro differentiating into keratinocyte-like cells. NETGIODS： The study was performed at the Academy of Military Medical Sciences from June 2005 to March 2006. The adipose tissues were obtained from patients ranged from 20 to 45 years （approved by them） by liposuction. The ADMSCs were isolated and cultured as described previous study by Zuk. Flow cytometry was performed in passage 3 cells to determine cell surface molecule and cell cycle. Passage 20 cells with well growth and fused to bottom were collected. Dividing cells were collected after 5 mg/L colchicines and performed karyotype analysis by Giemsa staining. The passage 3 stem cells were cultured under the medium with 5 μm tretinoin （ATRA） and 5μg/L epidermal growth factor （EGF） and then induced to differentiate into keratinocytes. ADMSCs in the control group were cultured in L-DNMEM medium with fetal calf serum （FCS） of 0.05 volume fraction. Cell morphology was observed under inverted microscope and CK19 expression was detected by immunohistochemical method. RESULTS：①ADMSCs expressed CD29, CD44 and CD90, and did not express CD34. ②Cell proliferative curve was ＂S＂ shape. An initial lag time of 3 days was observed, ADMSCs then entered a logarithmic phase, 6 days later entered platform phase. Population doubling time was about 29 hours. 80% cells in logarithmic phase were in Go and G1 phases. ③Analysis of cell chromosomal karyotypes showed that number of chromosome was 2n =46, 46, xx, normal female chromosomal karyotypes. ④The ADMSCs exhibited a fibroblast-like morphology, arranged in whirlpool shape and well adherence in the control group. ADMSCs in the test group from the 3rd after induction became irregular, after 10 days it

关 键 词：脂肪抽吸物 间充质干细胞 细胞分化 角质样细胞 

分 类 号：R394.2[医药卫生—医学遗传学]