中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (24): 4398-4401.doi: 10.3969/j.issn.1673-8225.2010.24.007

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

酶消化及差速贴壁法原代培养新生C57小鼠耳蜗膜蜗管外侧壁成纤维细胞

王庭阔，孙  虹   

  1. 中南大学湘雅三医院耳鼻咽喉-头颈外科，湖南省长沙市  410013
  • 出版日期:2010-06-11 发布日期:2010-06-11
  • 通讯作者: 孙 虹，博士，教授，中南大学湘雅三医院耳鼻咽喉-头颈外科，湖南省长沙市 410013 shjhaj@vip163.com
  • 作者简介:王庭阔☆，男，1971年生，湖南省沅陵县人，土家族，中南大学湘雅三医院在读博士，主治医师，主要从事耳科临床方面的研究。 wtk711112@163.com
  • 基金资助:

    国家自然科学基金(30371531)，课题名称“耳蜗兴奋性损伤后神经营养素-3对神经元的保护作用研究”；湖南省自然科学基金(02JJY2050)，课题名称“神经营养素-3和尼莫地平对抗耳蜗兴奋性损害的实验研究”；湖南省卫生厅科研基金(B2006-063)，课题名称“羟基磷灰石纳米载体介导神经营养素3基因治疗神经性耳聋”；湖南省科技厅科技计划重点项目(2007SK2001)，羟基磷灰石纳米载体介导内耳基因转染的关键技术研究。

Primary culture of fibroblasts from cochlear duct membrane of neonatal C57 mouse using enzyme digestion and differential adhesion methods  

Wang Ting-kuo, Sun Hong   

  1. Department of Otorhinolaryngology-Head & Neck Surgery, Third Xiangya Hospital, Central South University, Changsha  410013, Hunan Province, China
  • Online:2010-06-11 Published:2010-06-11
  • Contact: Sun Hong, Doctor, Professor, Department of Otorhinolaryngology-Head & Neck Surgery, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan Province, China shjhaj@vip163.com
  • About author:Wang Ting-kuo☆, Studying for doctorate, Attending physician, Department of Otorhinolaryngology-Head & Neck Surgery, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan Province, China wtk711112@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30371531*; the Natural Science Foundation of Hunan Province, No. 02JJY2050*; the Scientific Research Fund of Health Department of Hunan Province, No. B2006-063*; the Science and Technology Planning Project of Hunan Province, No. 2007SK2001*

PDF

451

被引次数

5

摘要：

背景：目前对耳蜗膜蜗管外侧壁成纤维细胞的培养没有特定的方法。
目的：采用酶消化与差速贴壁相结合的方法，原代培养新生C57小鼠膜蜗管外侧壁成纤维细胞，提供良好的体外细胞模型。
方法：显微解剖分离新生C57小鼠膜蜗管外侧壁组织，对膜蜗管外侧壁组织进行胰蛋白酶消化与差速贴壁相结合的方法培养成纤维细胞，倒置相差显微镜下观察细胞生长状态，苏木精-伊红染色，绘制细胞生长曲线，免疫组织化学染色鉴别细胞来源。
结果与结论：膜蜗管外侧壁组织来源传代纯化的成纤维细胞呈梭形和三角形，生长曲线成S形，免疫组织化学检测波形蛋白，细胞胞浆中呈棕黄色阳性反应。结果证实培养出小鼠膜蜗管外侧壁成纤维细胞。

关键词: 血迷路屏障, 离子通道, 细胞模型, 原代培养, 小鼠, 成纤维细胞

Abstract:

BACKGROUND: There has been no specific method for culturing fibroblasts from cochlear duct membranes so far.
OBJECTIVE: To provide good cell model in vitro via culturing fibroblasts from lateral wall of cochlear duct membrane of neonatal C57 mouse by enzyme digestion and differential adhesion methods and to identify its biological features.
METHODS: Tissues from lateral wall of cochlear duct membrane were acquired from neonatal C57 mouse using operating microscope. Fibroblasts from lateral wall of cochlear duct membrane were cultured by trypsin digestion combined with differential adhesion methods. The growth condition was observed by inverted phase contrast microscope and hematoxylin-eosin staining, and cell growth curve was drawn, then the immunocytochemistry was employed to classify cell types.
RESULTS AND CONCLUSION: After passage and purification, the shape of fibroblasts from lateral wall of cochlear duct membrane was fusiform and triangle, with “S” type cell growth curve. Immunocytochemiscal detection showed that vimentin could be detected in cultured cells, and brown cytoplasmic pigment could be seen. The results demonstrated that fibroblasts can be cultured form lateral wall of cochlear duct membrane of mice. 

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