构建组织工程化泪道上皮的潜在候选细胞：一项在兔身上的组织学和细胞学研究

Potential candidate cells for constructing tissue-engineered lacrimal duct epithelium: a histological and cytological study in rabbits.

作者信息

Xie Chen, Li Xiu-yi, Cui Hong-guang

机构信息

Department of Ophthalmology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.

出版信息

J Zhejiang Univ Sci B. 2015 Nov;16(11):904-13. doi: 10.1631/jzus.B1500113.

DOI:10.1631/jzus.B1500113

PMID:26537208

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4642871/

Abstract

OBJECTIVE

Injury and deficiency of the lacrimal duct epithelium (LDE) can lead to a variety of lacrimal diseases. The purpose of this study was to characterize potential candidate cells for constructing a tissue-engineered LDE.

METHODS

Different areas of the conjunctiva and lacrimal duct tissue were removed from male adult New Zealand white rabbits for histological evaluation. Hematoxylin and eosin staining and immunohistochemical staining of cytokeratin AE1+AE3, cytokeratin 4, Ki-67, and MUC5AC were observed by light microscopy. The surface morphologies of different epithelial tissues and cellular structures were examined using field-emission scanning electron microscopy and transmission electron microscopy. Epithelial cells were isolated from tissues and identified by specific markers. In vitro, proliferative ability and Western blot analyses of the proliferating cell nuclear antigen (PCNA) of different epithelial cells cultured in identical environments were investigated and compared.

RESULTS

Histologically, the epithelial specific markers, cytokeratin AE1+AE3 and cytokeratin 4, were expressed in the conjunctiva epithelium and the LDE. Notably, highly proliferative cells stained with Ki-67 were concentrated under the epithelium in a dome structure of the posterior palpebral conjunctiva. Differentiated goblet cells were also found to a lesser extent in this region. Primary palpebral and fornical conjunctival epithelial cells (PFCECs), bulbar conjunctival epithelial cells (BCECs), and lacrimal duct epithelial cells (LDECs) were successfully separated from tissues. In vitro, rabbit PFCECs and LDECs grew faster and expressed more PCNA than BCECs.

CONCLUSIONS

PFCECs are anatomically similar to LDECs. They also have similar morphological characteristics, immune phenotypes, and proliferation features. PFCECs are therefore potential candidate cells to replace LDECs in tissue engineering to treat lacrimal duct diseases.

摘要

目的

泪道上皮（LDE）的损伤和缺陷可导致多种泪道疾病。本研究的目的是鉴定用于构建组织工程化LDE的潜在候选细胞。

方法

从成年雄性新西兰白兔身上取下结膜和泪道组织的不同区域进行组织学评估。通过苏木精-伊红染色以及细胞角蛋白AE1+AE3、细胞角蛋白4、Ki-67和MUC5AC的免疫组织化学染色，在光学显微镜下进行观察。使用场发射扫描电子显微镜和透射电子显微镜检查不同上皮组织的表面形态和细胞结构。从组织中分离上皮细胞并通过特异性标志物进行鉴定。在体外，研究并比较在相同环境中培养的不同上皮细胞的增殖能力以及增殖细胞核抗原（PCNA）的蛋白质免疫印迹分析结果。

结果

组织学上，上皮特异性标志物细胞角蛋白AE1+AE3和细胞角蛋白4在结膜上皮和LDE中表达。值得注意的是，用Ki-67染色的高增殖细胞集中在上皮下方睑结膜后穹窿的圆顶结构中。在该区域也发现了少量分化的杯状细胞。成功从组织中分离出原发性睑结膜和穹窿结膜上皮细胞（PFCECs）、球结膜上皮细胞（BCECs）和泪道上皮细胞（LDECs）。在体外，兔PFCECs和LDECs比BCECs生长更快且PCNA表达更多。