红细胞裂解液法体外分离培养兔骨髓间充质干细胞  

Red blood cell lysis isolation and culture of rabbit bone marrow mesenchymal stem cells in vitro

作　　者：张卫东 章方彪 史宏灿 谭荣邦 叶钢 李广宇 潘枢 孙飞 

机构地区：[1]扬州大学临床医学院,江苏省扬州市225001

出　　处：《中国组织工程研究》2013年第49期8468-8473,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(30672080;30972968;81170014);江苏省"六大人才高峰"高层次人才基金(2009128);江苏省"青蓝工程"学术带头人基金(20081215)资助~~

摘　　要：背景:有研究向骨髓液中加入红细胞裂解液来提高分离纯化骨髓间充质干细胞的效率,以期获得纯度高、数量多的骨髓间充质干细胞。目的:进一步验证红细胞裂解液法体外分离纯化兔骨髓间充质干细胞的增殖特性,并对其进行细胞生物学鉴定。方法:使用红细胞裂解液提取含有兔骨髓间充质干细胞的骨髓悬混液,于4,7,10,13 d记录比较原代细胞的生长相对数量;观察传代细胞的生长情况,绘出P2、P3、P4及P5代细胞的生长曲线,比较各代细胞的增殖特点;倒置相差显微镜观察细胞的形态,细胞免疫荧光及流式细胞仪检测所获得细胞的CD44、CD34表面抗原的表达情况。结果与结论:红细胞裂解液法所获得的兔骨髓间充质干细胞为均一规则的以梭形为主的贴壁细胞,其胞核胞核均质、核仁明显、胞浆丰富,且CD44抗原表达阳性、CD34抗原表达阴性;原代细胞生长曲线呈类"S"型;P4代细胞具有较好的增殖活性,其生长速度、所获取的细胞数量均优于其他代数细胞。说明红细胞裂解液法可以在体外较好的培养出骨髓间充质干细胞,其P4代细胞是骨髓间充质干细胞增殖能力最强的一代细胞。BACKGROUND：Some studies have demonstrated that red blood celllysate added into the bone marrow can increase the efficiency of isolating and purifying mesenchymal stem cells, in order to obtain high-purity and high-quantity bone marrow mesenchymal stem cells. OBJECTIVE：To isolate and culture bone marrow mesenchymal stem cells of rabbit with red blood celllysis in vitro for exploring proliferation characteristics and performing the biological identification of cells. METHODS：Bone marrow suspension was col ected by puncturing the tibia with medul o-puncture needle. Red blood celllysis was added to the bone marrow suspension, and bone marrow mesenchymal stem cells were isolated and cultured. Numbers of primary cells were recorded at 4, 7, 10, and 13 days later And Growth curves of the cells at passages 2-5 were drawn for comparison of proliferative characteristics. Inverted phase contrast microscope was used to observe the morphological changes of cells. CD34, CD44 antigens of bone marrow mesenchymal stem cells were identified by flow cytometry and immunity fluorescence. RESULTS AND CONCLUSION：The adherent bone marrow mesenchymal stem cells mainly showed s spindle shape, with homogeneous nuclei, prominent nucleoli, and rich cytoplasm, which were positive for CD44 antigen and negative for CD34 antigen. The primary cells exhibited an“S”shape. Passage 4 cells had a better proliferative ability, rapider growth and more counting of cells as compared with other generations. These findings indicate that red blood celllysis method is a feasible ways of culturing bone marrow mesenchymal stem cells 0 in vitro, and passage 4 cells have the strongest proliferation capacity.

关 键 词：干细胞 骨髓干细胞 骨髓间充质干细胞 红细胞裂解液 细胞培养 分离纯化 细胞增殖 细胞生物学 国家自然科学基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]