中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (1): 94-99.doi: 10.3969/j.issn.2095-4344.2014.01.016

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

活体及体外条件下对角膜上皮干细胞的定位

许中中1，2，余晓菲2，杜连心2，李  晶2，王丽娅2   

  1. 1郑州大学第一附属医院眼科，河南省郑州市  450052
    2河南省眼科研究所，河南省郑州市  450003
  • 修回日期:2013-11-11 出版日期:2014-01-01 发布日期:2014-01-01
  • 通讯作者: 王丽娅，教授，博士生导师，河南省眼科研究所，河南省郑州市 450003
  • 作者简介:许中中，男，1982年生，河南省郑州市人，汉族，郑州大学第一附属医院在读博士，主治医师，主要从事角膜上皮干细胞的体外分离及培养、组织工程技术构建角膜缘上皮片及眼表重建方面研究。
  • 基金资助:

    国家自然科学基金(81170831)

Location of corneal epithelial stem cells under in vivo and in vitro conditions

Xu Zhong-zhong1, 2, Yu Xiao-fei2, Du Lian-xin2, Li Jing2, Wang Li-ya2   

  1. 1Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
    2Henan Eye Institute, Zhengzhou 450003, Henan Province, China
  • Revised:2013-11-11 Online:2014-01-01 Published:2014-01-01
  • Contact: Wang Li-ya, Professor, Doctoral supervisor, Henan Eye Institute, Zhengzhou 450003, Henan Province, China
  • About author:Xu Zhong-zhong, Studying for doctorate, Attending physician, Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China; Henan Eye Institute, Zhengzhou 450003, Henan Province, Chi
  • Supported by:

    the National Natural Science Foundation of China, No. 81170831

PDF

461

被引次数

12

摘要：

背景：眼表存在两种形式的上皮干细胞即角膜上皮干细胞和结膜上皮干细胞，角膜上皮干细胞在角膜上皮细胞更新和角膜透明的维持方面起着重要作用。
目的：采用活体激光扫描角膜共焦显微镜和免疫荧光染色技术相结合的方法，从活体和体外层面上对角膜上皮干细胞进行定位研究。
方法：收集2009年9月至2012年9月来河南省眼科研究所就诊的单侧角膜缘干细胞缺乏患者，使用活体激光扫描角膜共焦显微镜检查患者双眼，健侧眼为对照。扫描方位依次为中央角膜及上、下、左、右方的角膜缘，记录扫描图像并分析。眼球材料来自于河南省眼库，切取角膜中央和角膜缘组织，组织包埋剂包被、冰冻切片，切片厚度5-7 μm；免疫荧光染色技术检测p63、ABCG2、K3和Connexin 43在角膜中央及角膜缘上皮层的表达。
结果与结论：共有24例患者确诊为单侧角膜缘干细胞缺乏，活体激光扫描角膜共焦显微镜下患侧眼角膜病变区可见结膜细胞及杯状细胞；角膜缘区域Vogt栅栏状结构消失，色素细胞消失，取而代之的是大量纤维瘢痕化组织。免疫荧光染色示表达ABCG2和p63的细胞主要在角膜缘上皮基底层，尤其在近结膜侧的角膜缘及角膜缘中间部表达相对较高，而中央角膜上皮层细胞不表达；K3及Connexin43在角膜缘上皮基底细胞层不表达，中央角膜上皮全层表达。通过活体激光扫描角膜共焦显微镜观察及干细胞标记物检测显示角膜上皮干细胞主要存在于角膜缘外2/3区域的Vogt栅栏基底部及钉突结构中。


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, 培养, 角膜上皮干细胞, 活体激光扫描角膜共焦显微镜, 免疫荧光染色, 定位, 国家自然科学基金

Abstract:

BACKGROUND: There are two types of epithelial stem cells in the ocular surface tissue: corneal epithelial stem cells and conjunctival epithelial stem cells. The corneal epithelial stem cells play an important role in renewal of corneal epithelial cells and maintenance of corneal transparency.
OBJECTIVE: To study the location of corneal epithelial stem cells using laser in vivo confocal microscopy and immunofluorescent staining.
METHODS: Patients with unilateral limbal stem cell deficiency who went to Henan Eye Institute from September 2009 to September 2012 were enrolled in this study. Bilateral eyes were scanned by laser in vivo confocal microscopy, and the healthy eye was imaged as a control. The central cornea and limbus were scanned and images were recorded for statistical analysis. The eye balls were obtained from Henan Eye Bank, China. Central cornea and limbus were dissected and embedded in the OCT compound for frozen section and the proper thickness of the section was 5-7 μm. Immunofluorescent staining was used to detect the expression of p63, ABCG2, K3 and Connexin 43 in the epithelial layers of central cornea and limbus.
RESULTS AND CONCLUSION: Twenty-four patients diagnosed with unilateral limbal stem cell deficiency were 
recruited. Under confocal microscopy, in the affected eyes, the typical morphology of conjunctival epithelial cells and goblet cells was detected instead of corneal epithelial cells; in the limbus, a great amount of fiber scarring tissue was detected instead of Vogt palisade, rete pegs and pigment cells. Immunofluorescent staining showed the expression of p63, ABCG2 was mainly in the basal layer of limbal epithelium, especially in the outer and middle parts, but the expression of p63 and ABCG2 was not detected in the epithelial cell layers of central cornea. K3 and Connexin43 were not expressed in suprabasal layers of limbal epithelium, but in central cornea, they were expressed highly in the whole epithelial cell layers. Laser in vivo confocal microscopy and immunofluorescent staining showed the corneal epithelial stem cells were located in the basal layer of outer and middle limbal epithelium, mainly in Vogt palisade and rete pegs.


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, epithelium, corneal, microscopy, confocal, fluoroimmunoassay

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