密度梯度离心与贴壁筛选法体外分离培养胎儿骨髓间充质干细胞  

In vitro isolation and culture of fetal bone marrow mesenchymal stem cells using density gradient centrifugation combined with adherence screening method

作　　者：刘斌[1] 戎利民[1] 董健文[1] 曾春[2] 蔡道章[2] 

机构地区：[1]中山大学附属第三医院脊柱外科,广东省广州市510630 [2]中山大学附属第三医院关节外科 创伤骨科,广东省广州市510630

出　　处：《中国组织工程研究与临床康复》2009年第40期7913-7918,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省科技计划项目(2007B031001004,2005B34001003)~~

摘　　要：背景:胎儿骨髓间充质干细胞与成人相比,具有更好的自我更新和分化能力,以及更低的免疫原性。目的:选择更适应胎儿骨髓间充质干细胞体外分离培养的方法。设计、时间及地点:单一样本细胞学观察,于2005-06/12在中山大学附属第三医院中心实验室完成。材料:16～24周胎龄的流产胎儿由中山大学附属第三医院妇产科提供,产妇及家属均签署知情同意书。方法:无菌条件下截取流产胎儿双侧四肢骨,PBS冲洗髓腔,离心去脂肪及上清液,L-DMEM重悬细胞,沿管壁缓慢滴加至底部有等量1.073g/mLPercoll分离液的离心管中,离心后吸取中间界面白膜层,加入含体积分数为10%的胎牛血清、青霉素钠、链霉素的L-DMEM完全培养基,接种后置于37℃、体积分数为5%的CO2饱和湿度孵箱内培养。48h后换液,去除未贴壁细胞,以后每2d换液一次。待细胞达到80%～90%融合时,胰酶-EDTA消化传代。主要观察指标:倒置显微镜及Giemsa染色观察细胞形态;透射电镜观察细胞超微结构;流式细胞技术检测其表面标记物;碱性磷酸酶染色检测成骨分化能力;Ⅱ型胶原免疫细胞化学染色检测成软骨分化能力。结果:原代及传代培养的胎儿骨髓间充质干细胞呈梭形外观,具有较强的增殖能力。胞体较小,细胞核/浆比例大,胞核呈圆形或椭圆形,胞浆少,染色质较疏松,表现出早期细胞的特点,传至第20代细胞超微结构仍无明显改变。细胞表面抗原CD29,CD44,CD105呈阳性表达,CD34,CD45呈阴性表达。骨髓间充质干细胞诱导分化后,碱性磷酸酶染色及Ⅱ型胶原均呈阳性。结论:利用密度梯度离心+贴壁筛选法可成功分离培养胎儿骨髓间充质干细胞,所获细胞能够向成骨细胞及软骨细胞分化,该方法为体外培养扩增胎儿骨髓间充质干细胞的简单、稳定、高效的手段。BACKGROUND：Compared to adult bone marrow mesenchymal stem cells（MSCs） ,fetal MSCs has better self-renewal and differentiation potential,with lower immunogenicity. OBJECTIVE：To investigate in vitro culture and isolation method for fetal MSCs. DESIGN,TIME AND SETTING：A single sample cytology observation. The experiment was conducted at the Centre Laboratory of Third Affiliated Hospital,Sun Yat-sen University,from June to December 2005. MATERIALS：Aborted fetuses with 16-24 weeks were provided by Department of Gynaecology and Obstetrics,Third Affiliated Hospital of Sun Yat-sen University. The puerperant and her relatives all signed informed consents. METHODS：Bone of limbs were obtained under sterile condition,washed by PBS,centrifugalization,and dropwise into centrifuge tube containing 1.073 g/mL Percoll separating medium,centrifugalization,and then adding L-DMEM containing 10% fetal bovine serum,benzylpenicillin sodium and phytomycin,cultured in a incubator at 37 ℃ with 5% saturated humidity. The culture medium was renewed after 48 hours,and non-adhesive cells were removed. When the cells reached 80%-90% confluency,the digestion was terminated with addition of trypsin-EDTA and adhesive cells were passaged. MAIN OUTCOME MEASURES：The cell morphology was observed by inverted microscope and Giemsa staining;the ultrastructure of cells was detected by transmission electron microscope（TEM） ;flow cytometry was applied to detect the surface antigens. Expression of alkaline phosphatase was measured by alkaline phosphatase staining;moreover,the cartilage differentiation potency was determined by immunocytochemistry. RESULTS：The MSCs appeared morphologically spindle-shaped and presented active proliferative capacity in primary and passage cultures. The cellular nucleus showed round or oval shaped and the chromatin was loosened,with increased nuclear/cytoplasmic ratio. All of the above demonstrated the characteristics of early stage cells. Flow cytometry analysis indicated that fetal MSCs were universally

关 键 词：胎儿 骨髓间充质干细胞 培养 

分 类 号：R394.2[医药卫生—医学遗传学]