中国组织工程研究

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (49): 8468-8473.doi: 10.3969/j.issn.2095-4344.2013.49.002

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

红细胞裂解液法体外分离培养兔骨髓间充质干细胞

张卫东，章方彪，史宏灿，谭荣邦，叶  钢，李广宇，潘  枢，孙  飞   

  1. 扬州大学临床医学院，江苏省扬州市  225001
  • 修回日期:2013-09-14 出版日期:2013-12-03 发布日期:2013-12-03
  • 通讯作者: 通讯作者：史宏灿，博士，主任医师，博士生导师，扬州大学临床医学院，江苏省扬州市 225001
  • 作者简介:张卫东★，男，1988年生，河南省驻马店市人，汉族，扬州大学在读硕士，主要从事组织工程气管构建的研究。 Zhangw20062008@126.com
  • 基金资助:

    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*

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

Zhang Wei-dong, Zhang Fang-biao, Shi Hong-can, Tan Rong-bang, Ye Gang, Li Guang-yu, Pan Shu, Sun Fei   

  1. Medical College of Yangzhou University, Yangzhou  225001, Jiangsu Province, China
  • Revised:2013-09-14 Online:2013-12-03 Published:2013-12-03
  • Contact: Shi Hong-can, M.D., Chief physician, Doctoral supervisor, Medical College of Yangzhou University, Yangzhou 225001, Jiangsu Province, China shihongcan@hotmail.com
  • About author:Zhang Wei-dong★, Studying for master’s degree, Medical College of Yangzhou University, Yangzhou 225001, Jiangsu Province, China Zhangw20062008@126.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30672080*, 30972968*, 81170014*; the High-level Personnel Fund for the “Six Talents Peak” of Jiangsu Province, No. 2009128*; Jiangsu Province “Blue Project” Academic Leaders Fund, No. 20081215*

PDF

777

被引次数

8

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。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

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

Abstract:

BACKGROUND: Some studies have demonstrated that red blood cell lysate 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 cell lysis in vitro for exploring proliferation characteristics and performing the biological identification of cells. 
METHODS: Bone marrow suspension was collected by puncturing the tibia with medullo-puncture needle. Red blood cell lysis 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 cell lysis method is a feasible ways of culturing bone marrow mesenchymal stem cells 0 in vitro, and passage 4 cells have the strongest proliferation capacity.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

Key words: stem cells, mesenchymal stem cells, cells, cultured, cell proliferation

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