中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (24): 4491-4494.doi: 10.3969/j.issn.1673-8225.2012.24.025

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

组织块法培养成年兔肌腱细胞

杨 光1，姜 涛2，王振兴1，张 巨1   

  1. 吉林大学中日联谊医院，1手外科，2血管外科，吉林省长春市 130033
  • 收稿日期:2011-11-15 修回日期:2011-12-15 出版日期:2012-06-10 发布日期:2013-11-05
  • 通讯作者: 张巨，教授，硕士生导师，吉林大学中日联谊医院手外科，吉林省长春市 130033
  • 作者简介:杨光☆，男，1978 年生，吉林省通榆县人，汉族，2007年吉林大学毕业，博士，主治医师，主要从事肌腱和周围神经损伤修复研究。 yangguang611@163.com
  • 基金资助:

    吉林省卫生厅基金课题资助项目(20082050)。课题名称为“血小板源性生长因子对肌腱愈合影响的实验研究”

Culture of adult rabbit tenocytes using tissue explant method

Yang Guang1, Jiang Tao2, Wang Zhen-xing1, Zhang Ju1   

  1. 1Department of Hand Surgery, 2Department of Vascular Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin Province, China
  • Received:2011-11-15 Revised:2011-12-15 Online:2012-06-10 Published:2013-11-05
  • Contact: Zhang Ju, Professor, Master’s supervisor, Department of Hand Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin Province, China
  • About author:Yang Guang☆, Doctor, Attending physician, Department of Hand Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin Province, China yangguang611@163.com

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摘要：

背景：体外分离培养肌腱细胞，熟悉其生物学特性是研究肌腱愈合机制、改善肌腱愈合内环境的前提和基础。
目的：采用组织块法体外培养成年兔肌腱细胞，观察其细胞形态、生长增殖情况以及Ⅰ型、Ⅲ型胶原蛋白的表达。
方法：无菌条件下取成年新西兰白兔双侧后肢趾屈肌腱，手术显微镜下剥离、去除肌腱外膜组织，剪成组织小块，0.25%胰蛋白酶和0.1%胶原酶Ⅰ消化10~15 min，离心去上清，将组织小块转移到培养瓶中，贴壁后加入1 mL培养液，待细胞游出贴壁生长时，再加培养液继续培养，每3 d换液1次，当细胞长到80%~90%融合时按1∶3传代。
结果与结论：一般10 d左右细胞会从组织块游出贴壁生长，此时细胞多呈星形或不规则形，随着时间延长细胞逐渐增多，呈梭形的成纤维细胞样。传代细胞刚接种时圆形，4~6 h开始贴壁并伸展为梭形，排列逐渐规则成群。从传代细胞的生长曲线可看出，前4 d细胞生长较慢，为潜伏期，第5天后进入快速增殖期，7 d以后进入平台期。免疫荧光法证实Ⅰ型胶原染色阳性，而Ⅲ型胶原染色呈阴性。结果表明采用组织块法可在体外成功培养成年兔肌腱细胞。

关键词: 肌腱细胞, 兔, 生物学性状, 分离, 组织块法, 组织构建

Abstract:

BACKGROUND: Culture in vitro and understanding the biological characteristics of tenocytes are the premise and foundation to study the mechanism and improve the internal environment of tendon healing.
OBJECTIVE: To culture adult rabbit tenocytes in vitro with tissue explant, investigate the morphology, growth and proliferation of tenocytes, and to test the expression of collagen Ⅰ and collagen Ⅲ in the cells.
METHODS: After adult New Zealand rabbit flexor tendon was obtained under aseptic condition, the peritenon of tendon was removed. The tendon was divided into small fragments. The fragments were digested with 0.25% trypsin and 0.1% collagenaseⅠfor 10-15 minutes. The fragments were transferred into culture flasks after centrifugation. And 1 mL culture medium was added into the flasks after the fragments attaching to the wall. Culture medium was added when the cells showed an adhesive growth, and the medium was replaced every 3 days. When 80%-90% of the cells were in confluence, they were passaged at a ratio of 1:3.
RESULTS AND CONCLUSION: The tenocytes showed an adhesive growth at 10 days, and appearance was star-shaped or irregular shaped. The number of tenocytes was increased and the cell appearance changed to fibroblast-like as went on. Passaged cells were round-shaped at the beginning of cell seeding, the cells attached to the wall after 4-6 hours showed a spindle-shape, and the cells gradually arranged in groups. The growth curve of passage cells showed that: the latent period was the first 4 days, the logarithm period was at 5-6 days, and the platform period was at 7 days. Type Ⅰ collagen was positive and type III collagen was negative tested by immunofluorescence assay. The results indicated that tenocytes can be successfully isolated and cultured from adult rabbit tendon in vitro with tissue explant.

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