人骨髓间充质干细胞的分离培养及BrdU标记鉴定  

Separation,culture and BrdU labeling of human bone marrow mesenchymal stem cells

作　　者：张向荣[1] 郭光华[2] 刘德伍[2] 彭燕[2] 

机构地区：[1]南昌大学第一附属医院眼科,江西省南昌市330006 [2]南昌大学第一附属医院烧伤中心,江西省南昌市330006

出　　处：《中国组织工程研究与临床康复》2009年第19期3618-3622,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江西省科技厅重大科技招标项目(200604);江西省卫生厅重大科技招标项目(200502);江西省自然科学基金资助项目(0540056)~~

摘　　要：背景:骨髓间充质干细胞具有多向分化能力,是目前极具潜力的细胞及基因治疗的靶细胞。目的:拟在体外建立一种分离纯化、培养扩增、标记骨髓间充质干细胞的方法。设计、时间和地点:开放性实验,于2008-03/05在南昌大学第一附属医院烧伤研究所完成。材料:人骨髓间充质干细胞取自临床骨髓穿刺检查正常的住院患者,无血液系统疾病和家族遗传病史,由南昌大学第一附属医院提供。方法:密度梯度离心和贴壁培养相结合,分离纯化骨髓间充质干细胞,并用BrdU标记。主要观察指标:相差显微镜下观察人骨髓间充质干细胞的形态变化;流式细胞仪检测人骨髓间充质干细胞的表面标记以及细胞周期;绘制人骨髓间充质干细胞生长曲线;透射电镜观察人骨髓间充质干细胞的超微结构。结果:密度梯度离心结合贴壁法能分离培养出纯度较高的人骨髓间充质干细胞。流式细胞仪检测人骨髓间充质干细胞CD90,CD29,CD44,CD34,CD45阳性细胞表达分别为98.48%,98.74%,97.41%,0.36%,0.64%。人骨髓间充质干细胞的生长曲线呈S形。透射电镜可见人骨髓间充质干细胞胞质丰富,粗面内质网发达,大量蛋白分泌物。免疫组织化学检测BrdU标记48h后人骨髓间充质干细胞标记率>80%。结论:采用淋巴细胞分离液密度梯度离心和贴壁培养相结合,以获得纯度较高和活性骨髓间充质干细胞,是简便可行的方法;BrdU是一种简单、有效的标记骨髓间充质干细胞的方法。BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are capable of multiple directional differentiation, and are potential cells and target cells for gene therapy. OBJECTIVE： To establish BMSC isolation, purified, cultured, amplified, labeled method in vitro. DESIGN, TIME AND SETTING： An open experiment was performed at the Institute of Burns of First Affiliated Hospital of Nanchang University between March and May 2008. MATERIALS： Human BMSCs were harvested from bone marrow of patients without the blood systemic diseases or familial inheritance disease history in clinical examination at the First Affiliated Hospital of Nanchang University. METHODS： BMSCs were isolated by combining density gradient centrifugation with plastic adherence, and then labeled by BrdU. MAIN OUTCOME MEASURES： Morphological observations were performed with phase contrast microscope; growth curves of the cells were drawn by cytometry method; cell phenotype and generation cycle were detected by flow cytometry; BMSC ultrastructures were determined by transmission electron microscope. RESULTS： Higher purity of human BMSCs could be achieved by the density gradient centrifugation. The positive expression rates of cell phenotypes were various as followings respectively： CD90, 98.48%; CD29, 98.74%; CD44, 97.41%; CD34, 0.36%; CD45, 0.64%. The growth curve of BMSCs was ＂S＂ shape. Abundant cytoplasm, prosperous rough endoplasmic reticulum and plenty of protein excretion were found inside BMSCs under transmission electron microscopes. The positive rate of BMSCs labeled by BrdU was above 80% after 48 hours detected using immunohistochemistry. CONCLUSION： Higher purity of BMSCs can be isolated and cultured by combining density gradient centrifugation with adherence Labeling BMSCs with BrdU is a simple efficient labeled method.

关 键 词：骨髓间充质干细胞 分离 培养 生物学鉴定 5-溴脱氧尿嘧啶核苷 

分 类 号：R394.2[医药卫生—医学遗传学]