中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (50): 9366-.doi: 10.3969/j.issn.1673-8225.2010.50.014

• 组织构建实验造模 • 上一篇    下一篇

小鼠肺微血管内皮细胞磁珠分选法分离和原代培养

孙振朕1，蔡在龙2，朱科明1，邓小明1   

  1. 解放军第二军医大学长海医院，  1麻醉科， 2中心实验室，上海市  200433
  • 出版日期:2010-12-10 发布日期:2010-12-10
  • 通讯作者: 朱科明，教授，主任医师，解放军第二军医大学长海医院麻醉科，上海市 200433 kmzhu_md@yahoo.com.cn； 邓小明，教授，主任医师，解放军第二军医大学长海医院麻醉科，上海市 200433 xmdeng@anesthesia.org.cn
  • 作者简介:孙振朕☆，女，1979年生，山东省青岛市人，汉族，解放军第二军医大学在读博士，主治医师，主要从事脓毒症急性肺损伤方面的研究。 Zhenzhensun2008@hotmail.com
  • 基金资助:

    国家自然科学基金项目(30872459)，课题名称：脓毒症急性肺损伤时血小板TLR4在肺微血管内皮细胞损伤中的作用机制研究。

     

Magnetic beads isolation and primary culture of mouse pulmonary microvascular endothelial cells in vitro

Sun Zhen-zhen1, Cai Zai-long2, Zhu Ke-ming1, Deng Xiao-ming1   

  1. 1 Department of Anesthesiology, 2 Clinical Experimental Center, Changhai Hospital, Second Military Medical University of  Chinese PLA, Shanghai  200433,  China
  • Online:2010-12-10 Published:2010-12-10
  • Contact: Zhu Ke-ming, Professor, Chief physician, Department of Anesthesiology, Changhai Hospital, Second Military Medical University of Chinese PLA, Shanghai 200433, China kmzhu_md@yahoo.com.cn Deng Xiao-ming, Professor, Chief physician, Department of Anesthesiology, Changhai Hospital, Second Military Medical University of Chinese PLA, Shanghai 200433, China xmdeng@anesthesia.org.cn
  • About author:Sun Zhen-zhen☆, Studying for doctorate, Attending physician, Department of Anesthesiology, Changhai Hospital, Second Military Medical University of Chinese PLA, Shanghai 200433, China zhenzhensun2008@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30872459*

     

PDF

1170

被引次数

22

摘要：

背景：肺组织的功能依赖于肺微血管内皮细胞的活性，因此肺微血管内皮细胞是相关研究的重要细胞模型，但目前国内多采用的组织块贴壁法培养所得的肺微血管内皮细胞常有其他细胞混杂。

目的：建立一种有效分离、培养、扩增小鼠肺微血管内皮细胞的方法。

方法：采用酶消化、免疫磁珠二次分选法分离纯化小鼠肺微血管内皮细胞，贴壁培养法体外扩增，CCK-8法测定细胞的生长情况，相差显微镜观察培养细胞的形态，透射电子显微镜观察细胞的超微结构，流式细胞仪对其表型进行鉴定。

结果与结论：培养所得小鼠肺微血管内皮细胞具有典型的铺路石样形态学特征，含有大量内皮细胞特有的杆状细胞器Weibel-Palade小体，较稳定地表达内皮细胞特异性表面标记CD105，不表达淋巴管内皮细胞特异性表面标记血管内皮生长因子受体3。说明免疫磁珠二次分选法可成功分离纯化小鼠肺微血管内皮细胞，体外培养所获细胞纯度高、自我更新能力强，并保留了包括构成、表面抗原表达等特性。

关键词: 免疫磁珠, 分离, 小鼠, 肺微血管内皮细胞, 细胞培养

Abstract:

BACKGROUND: Lung function depends on the activity of pulmonary microvascular endothelial cells (PMVECs). It is a very important cell model in related researches, while the cells which are isolated by the tissue block culture adhesion method that are currently widely used in China have a major disadvantage of being usually mixed with other cells.
OBJECTIVE: To establish an accurate and effective technique and methods of isolation and culture mouse PMVECs in vitro.
METHODS: PMVECs were isolated and purified by collagenase I digesting and magnetic beads separating,and adherent cultured in vitro. The growth curve of cultured PMVECs was measured by CCK-8, the cell morphology and ultrastructure was observed by microscopes and its phenotype was identified by flow cytometry.
RESULTS AND CONCLUSION: The cells grew as a monolayer in culture and exhibited the well-described cobblestone morphology, contained a lot of endothelial cell specific rhabditiform organelles Weibel-Palade bodies, expressed endothelial cell specific surface marker CD105 relatively stably, and rarely expressed lymphatic endothelial cell specific surface marker VEGFR-3. The PMVECs isolated successfully by immunoglobulin magnetic beads are of high purity and rapid multiplication and retain certain of their functional differences, including constitution and antigen molecule expression.

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