中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (7): 1019-1024.doi: 10.3969/j.issn.2095-4344.2016.07.015

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

兔主动脉血管内皮细胞原代改良提取法

肖紫春，谭金海，周 益   

  1. 武汉大学中南医院骨科，湖北省武汉市 430071
  • 收稿日期:2016-01-04 出版日期:2016-02-12 发布日期:2016-02-12
  • 通讯作者: 谭金海，博士，主任医师，教授，硕士生导师，武汉大学中南医院骨科，湖北省武汉市 430071
  • 作者简介:肖紫春，男，1990年生，湖南省衡阳县人，汉族，武汉大学在读硕士，主要从事骨组织工程研究。
  • 基金资助:
    湖北省自然科学基金重点项目(2014CFA084)

Improved extraction of primary vascular endothelial cells from the rabbit aorta

Xiao Zi-chun, Tan Jin-hai, Zhou Yi   

  1. Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei Province, China
  • Received:2016-01-04 Online:2016-02-12 Published:2016-02-12
  • Contact: Tan Jin-hai, M.D., Chief physician, Professor, Master’s supervisor, Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei Province, China
  • About author:Xiao Zi-chun, Studying for master’s degree, Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei Province, China
  • Supported by:

    the Natural Science Foundation of Hubei Province, No. 2014CFA084

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被引次数

1

摘要：

文章快速阅读：

文题释义：

血管内皮细胞：很多研究中又称之为内皮细胞，一般是指衬于心脏、血管及淋巴管内表面，排列紧密，是一单层扁平上皮细胞，它与少量的成纤维细胞同形成血管的内壁，维持着血管结构和功能的完整性。它不仅具有吞噬细菌、异物及坏死物和衰老的组织功能，还具备参与集体免疫活动、分泌和支撑形成血管结构等多种功能。还在物理屏障及凝血过程中起着重要的作用。

原代细胞：是指从机体取出后立即培养的细胞。严格来说，是指没有成功传代之前的细胞，此时的细胞保持原有特性。但通常人们把培养的第1代细胞与第10代以内的细胞统称为原代细胞培养。最常用的原代培养有组织块培养和分散细胞培养。原代细胞不仅广泛应用于分子、细胞生物学和生物医学基础研究，如蛋白质组学、基因组学、细胞株(系)研究、DNA，RNA和遗传学研究等，还可应用于当今热门的生物医药产业如药物筛选、药物代谢和毒理研究、癌症药物的研究等。 

 

背景：目前血管内皮细胞原代获取方法中，使用较多的就是大动脉血管内皮细胞培养及微动脉血管内皮细胞培养，传统多采用酶消化法及组织贴壁法，所获得血管内皮细胞数量和纯度已经不能满足科研实验的需要。

目的：探讨兔主动脉血管内皮细胞的原代的改良提取及鉴定方法。
方法：切取一段兔主动脉，甲组采用改良提取法培养血管内皮细胞，显微器械剥离出血管内层，再用酶消化法获得单个原代细胞，以内皮细胞培养液培养；乙组采用组织贴壁法生长，整个血管内面贴于培养皿中，同时于体积分数5%CO2，37 ℃温箱中孵育1 h。分别获得2组细胞沉淀并进行体外培养。采用显微镜观察细胞形态，通过免疫组织化学染色检测CD31、Ⅷ因子及Vimentin蛋白进行血管内皮细胞鉴定，并与组织贴壁生长法培养对比。

结果与结论：采用改良提取法得出的内皮细胞纯度比组织贴壁生长高，数量更多。免疫组织化学鉴定结果CD31(+)，Ⅷ因子(+)，Vimentin蛋白(-)。结果表明此法可以更容易获得纯度较高，数量更多的血管内皮细胞，可操作性及实用性较强。 

ORCID: 0000-0001-7644-2970(谭金海)

关键词: 组织构建, 血管内皮细胞, 兔主动脉, 酶消化法, 改良提取, 湖北省自然科学基金

Abstract:

BACKGROUND: Primary vascular endothelial cells are mostly harvested through aorta endothelial cell cultures and micro-artery endothelial cell cultures using enzyme digestion and tissue adhesion methods, and the quality and purity of harvested cells cannot meet the need for current scientific research.
OBJECTIVE: To investigate an improved extraction of primary vascular endothelial cells and the relevant identification method.
METHODS: A segment of rabbit aorta was cut to culture vascular endothelial cells using the improved extraction method in group A or using adhesion method in group B. In the group A, the vascular intima was striped out with microsurgical instruments, and digested enzymatically to acquire single primary cells followed by culture in endothelial cell culture medium. In the group B, the whole vascular intima was adhered to the culture dish that was incubated in a 5%CO2, 37 ℃ incubator for 1 hour. Cell pellets in the two groups were cultured in vitro. Cell morphology was observed using a microscope; immunohistochemical staining was used to detect CD31, VIII factor and Vimentin protein for identification of vascular endothelial cells.
RESULTS AND CONCLUSION: The purity and number of vascular endothelial cells extracted by the improved method were higher than those by the adhesion method. Immunohistochemical findings showed positive expression of CD31 and VIII factor, but negative expression of Vimentin. These findings indicate that the improved extraction method can obtain more vascular endothelial cells with higher purity, which is of strong operability and practicality.