人羊膜修复病理眼表的实验研究

Experimental study on human amniotic membrane transplantation for ocular surface reconstruction

目的通过对人羊膜的实验室研究，明确人羊膜上皮不同位置细胞的干细胞活性差异，以指导临床医生取材、制备、保存和应用人羊膜植片。为病理眼表修复中羊膜植片的应用提供实验依据。方法对羊膜植片进行实验室检查，应用HE染色、免疫组织化学染色方法观察不同厚薄羊膜材料上皮细胞排列分布情况；应用胰酶消化分离羊膜上皮细胞，采用免疫组织化学法、流式细胞分析术、免疫荧光法测定人羊膜上皮近基底层细胞与近表层细胞表达胚胎干细胞标志物（SSEA-3、SSEA-4、SSEA-1、TRA1-60、TRA1-81）、多潜能细胞转录因子（Nanog、Oct4、Sox2）的差异性。结果人羊膜近脐带区较厚，约为0．4mm；人羊膜上皮近基底层细胞区域胚胎干细胞标志物（SSEA-3、SSEA-4、SSEA-1、TRA1-60、TRA1—81）和多潜能细胞转录因子（Nanog、Oct4、Sox2）表达阳性率明显高于人羊膜上皮近表层细胞区域，差异有统计学意义（P〈0．05）。结论羊膜上皮近基底层细胞区具有更强的类胚胎干细胞活性，临床进行人羊膜取材时，应尽量选用较厚的新鲜人羊膜组织进行制备和移植，更有利于眼表修复的成功。

Objective To investigate different activities of human stem ceils in difference places of human amniotic epithelial in the way of laboratory tests to guide the extraction, preparation, conservation and application of human amniotic membrane, and to provide the laboratory and clinical evidence for the method selection to repair the lesions of ocular surface. Methods The distribution of epithelial cells in dif- ferent thicknesses of amniotic membranes was observed by hematoxylin eosin （HE） stain and immunohisto- chemistry method. The cells of human amniotic membrane were isolated by trypsin digestion. The differenti- al expressions of Nanog, Oct4, Sox2, SSEA-3, SSEA-4, TRA1-60 and TRA1-81 in basal epithelial cells and superficial epithelial cells of human amniotic membrane were detected by immunohistochemistry, flow cytometry and immunofluorescence assay. The data were analyzed statistically. Results HE staining showed that the thicker part of the amniotic membrane nearby the umbilical cord was about 0.4 mm, which used for human amniotic membrane transplantation possessed the structure Of stratified epithelial cells of am- niotic membrane. However, the thinnest part of the amniotic membrane nearby the umbilical cord possessed the structure of simple epithelial cells, and the embryonic-stem-cell-like function areas of amniotic mem- brane mainly distributed in the epithelial cell layer of amniotic membrane. Immunohistochemistry, flow cy- tometry and immunofluorescence assay showed that the positive rate of the expressions of Nanog, Oct4, Sox2, SSEA-3, SSEA-4, TRA1-60 and TRA1-81 in basal epithelial cells of amniotic membrane were signifi- cantly lower than in superficial epithelial cells of amniotic membrane, with a statistical significance （ P 〈 0. 05）. It demonstrated that the basal epithelial cells of amniotic membrane had more embryonic-stem-cell- like activity. Conclusions The part of the amniotic membrane nearby the umbilical cord is thicker, whichis rich in epithelial cells. The embryonic-stem-cell-like function areas of amniotic membrane mainly distrib- ute in the epithelial cell layer. The basal epithelial cells of amniotic membrane have greater embryonic-stem- cell-like activity than superficial epithelial cells of amniotic membrane. Therefore, the thicker fresh human amniotic membrane should be chosen as far as possible for preparation and transplantation, which would be a more effective surgical method for the ocular surface repair successfully.