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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2015 Vol.16 No.11 P.904-913
Potential candidate cells for constructing tissue-engineered lacrimal duct epithelium: a histological and cytological study in rabbits
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.
Key words: Conjunctival epithelial cells, Cytokeratin, Lacrimal duct epithelial cells, Mucin, Proliferating cell nuclear antigen, Tissue engineering
创新点：首次在活体及离体水平上比较不同部位的结膜上皮细胞与泪道上皮细胞的生物组织特性的相似性，以获得构建组织工程泪道上皮的理想候选细胞。
方法：取雄性新英格兰大白兔的不同区域的结膜及泪道组织，用于组织生物学评价。采用苏木精-伊红染色法及免疫组织化学鉴定细胞角蛋白AE1+AE3、细胞角蛋白4、人细胞增殖细胞核核抗原Ki-67及黏蛋白MUC5AC（图1）。场发射扫描电镜及投射电镜观察不同的上皮表层形态及细胞结构（图2）。此外，用酶化法将上皮细胞从组织分离，并用特定的抗体鉴定细胞（图3）。比较不同区域来源的结膜上皮细胞及泪道上皮细胞的离体增殖能力和细胞表达的增殖细胞核核抗原（PCNA）的差异（图4）。
结论：新英格兰大白兔的睑结膜上皮细胞在组织结构上与泪道上皮细胞更加接近。它们拥有相似的形态特征、蛋白免疫表型及增殖活性。睑结膜部的结膜上皮细胞可能是潜在的候选细胞用于构建组织工程的泪道上皮，进而用于治疗泪道疾病。
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DOI:
10.1631/jzus.B1500113
CLC number:
R777.2; Q813
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On-line Access:
2015-11-04
Received:
2015-05-11
Revision Accepted:
2015-09-30
Crosschecked:
2015-10-20