成人骨髓间充质干细胞的鉴定与体外BrdU标记  

Identification and in vitro BrdU labeling of adult bone marrow mesenchymal stem cells

作　　者：王小蕊[1] 侯相麟[1] 席亚明[1] 张海红[1] 

机构地区：[1]兰州大学第一医院血液科,甘肃省兰州市730000

出　　处：《中国组织工程研究与临床康复》2008年第21期4131-4135,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：甘肃省科技厅自然科学基金项目(3ZS041-A25-058)~~

摘　　要：背景:目前尚未发现骨髓间充质干细胞独有的标志分子,给其鉴定带来一定困难。BrdU标记不存在放射性污染,有研究认为在不受到紫外线照射的条件下,BrdU标记后对细胞功能无损害。目的:探讨成人骨髓间充质干细胞体外鉴定和标记的方法,并观察其生物学特性。设计、时间及地点:细胞观察,于2007-06/12在解放军兰州军区兰州总医院完成。材料:成人骨髓来自5例健康志愿者。BrdU为美国Sigma公司产品。方法:采集成人骨髓10mL,密度梯度法分离单个核细胞,加入含10%胎牛血清的DMEM培养基,调整细胞密度为2×108L-1进行扩增培养。取第3代骨髓间充质干细胞,加入成骨诱导培养基,行碱性磷酸酶染色;加入成脂诱导培养基,行油红O染色;加入终浓度为5,10,15μmol/LBrdU溶液,孵育12,24,48,72,96h后行免疫细胞化学染色。主要观察指标:倒置光学显微镜下观察细胞生长及增殖情况,流式细胞仪鉴定细胞表型,成骨及成脂诱导分化结果。BrdU阳性标记率及标记后细胞生长情况。结果:①体外培养的成人骨髓间充质干细胞形态均一,为梭形或纺锤形的成纤维细胞样外观;表达CD44和CD71,不表达CD34和HLA-DR;可定向诱导分化为成骨细胞和脂肪细胞。②大部分骨髓间充质干细胞标记后核抗BrdU染色阳性,随着标记浓度的升高和标记时间的延长,BrdU阳性率逐渐增高,终浓度10μmol/LBrdU标记72h后阳性率达90%以上,且连续传9代均可检测到BrdU阳性细胞。第3代骨髓间充质干细胞90%处于G0/G1期,BrdU标记后88.62%处于G0/G1期,标记前后细胞生长周期无明显变化。结论:通过形态学特征、表面标记和多向分化潜能可以鉴定体外分离培养的细胞为成人骨髓间充质干细胞;BrdU标记可用于成人骨髓间充质干细胞移植入体内后存活、生长和分化的动态示踪观察;BrdU最佳标记浓度和时间分别为10μmol/L和72h。BACKGROUND： No particular marker molecule of bone marrow mesenchymal stem cells （BMSCs） is presently found, so its determination is difficult. BrdU marker has no radioactive pollution. Some studies have confirmed that BrdU marker has no damage to cell function without ultraviolet radiation. OBJECTIVE： To investigate in vitro identification and labeling methods and biological characteristics of adult BMSCs. DESIGN, TIME AND SETTING： The cell experiment was conducted at the General Hospital of Lanzhou Military Area Command of Chinese PLA between June and December 2007. MATERIALS： Bone marrow was collected from 5 healthy adult volunteers. BrdU was purchased from Sigma, USA. METHODS： 10 mL adult bone marrow was harvested to isolate mononuclear cells by density gradient. Cells were cultured in DMEM containing 10% fetal bovine serum and proliferated at a density of 2× 10^8 L^-1. At the third passage, BMSCs was inoculated in medium supplemented with osteoblast inductor and stained with alkaline phosphatase. Subsequently, BMSCs were inoculated in medium supplemented with lipoblast inductor and stained with oil red O. Cells were incubated with BrdU at different concentrations of 5, 10 and 15 μ mol/L for 12, 24, 48, 72 and 96 hours, and then detected by immunocytochemistry. MAIN OUTCOME MEASURES： Cell growth and proliferation were observed under an inverted light microscope. Cell phenotype osteoblast and lipoblast differentiation were identified by flow cytometry. BrdU-positive labeling rate and cell growth after labeling were investigated. RESULTS： In vitro cultured BMSCs were homogenous population and exhibited a spindle-shaped fibroblastic morphology. BMSCs expressed CD44 and CDTI, but did not express CD34 and HLA-DR. BMSCs can differentiate into osteoblasts and lipoblasts. Most BMSCs were labeled by BrdU. With the increase in labeling concentration and over time, BrdU positive rate gradually increased and exceeded 90% after labeling with BrdU （10 μ mol/L） for 72 hours, with the labe

关 键 词：骨髓间充质干细胞 诱导分化 BRDU 

分 类 号：R394.2[医药卫生—医学遗传学]