人眼睑组织来源的成纤维细胞作为饲养层细胞构建人口腔黏膜上皮细胞片层的研究  

Cultivation of human oral mucosal epidermal cell sheets using human eyelid tissue-derived feeder layers

作　　者：张明琦 王乐 孙岩 郑玉强[1,2] 李娜 王卓实 ZHANG Mingqi;WANG Le;SUN Yan;ZHENG Yuqiang;LI Na;WANG Zhuoshi(Stem Cell Center of Precison Medicine Innovation Institute,He University,Shenyang 110001,China;Shenyang He Eye Hospital;Liaoning Key Lab of Ophthalmic Stem Cells)

机构地区：[1]辽宁何氏医学院精准医疗研究院,辽宁沈阳110001 [2]沈阳何氏眼科医院 [3]辽宁省眼科干细胞组织工程重点实验室

出　　处：《沈阳医学院学报》2023年第2期127-130,共4页Journal of Shenyang Medical College

基　　金：沈阳市科技计划项目(No.20-301-4-00);辽宁省自然基金指导计划(No.2019-ZD-0995);辽宁省自然基金资助计划(No.2019-MS-257)。

摘　　要：目的:探讨人眼睑组织来源的成纤维细胞作为饲养层细胞代替鼠源饲养层细胞在体外培养人口腔黏膜上皮细胞的可行性。方法:通过酶消化法从人眼睑组织中分离成纤维细胞传代培养,取P5代成纤维细胞通过丝裂霉素C方法制备饲养层细胞,将其制备成的饲养层细胞用来培养商品化人口腔黏膜上皮细胞。通过罗丹明染色观察其细胞克隆形成率;HE染色观察细胞片层的结构;免疫荧光检测特征蛋白P63、β1-integrin的表达。结果:人眼睑组织来源的成纤维细胞在体外具有较强的增殖能力,高表达成纤维细胞特异性标志性蛋白CD90,培养的人口腔黏膜上皮细胞呈克隆样生长,细胞克隆形成率约为2.2%,并将其构建成3~4层口腔黏膜细胞片层,且高表达上皮细胞标志物P63及β1-integrin。结论:人眼睑组织来源的成纤维细胞可以作为饲养层细胞构建人口腔黏膜上皮细胞片层,为临床应用提供一种新的人源化饲养层工具细胞。Objective:To investigate the feasibility of human eyelid tissue-derived fibroblast cells as feeder layer instead of murine 3T3 fibroblast cells to support engineered human oral mucosal epithelial cell sheets(human-OECS).Methods:Human primary fibroblast cells were isolated from human eyelid tissue by enzymatic digestion and then characterized using CD90 antibody by flow cytometry.Human oral mucosal epithelial cells were cultured in the presence of mitomycin C-treated human fibroblasts feeder layer.The colony-forming efficiency(CFE)was detected by Rhodamine staining.The structure of human-OECS was observed by HE staining.The expressions of P63 and β1-integrin were detected by immunofluorescence.Results:It was found that human eyelid tissue-derived fibroblasts showed a high proliferative capacity,with fibroblast-like morphologies and high expression of CD90(a marker of fibroblasts).Stratified human-OECS with 3-4 layers of epidermal cells was successfully cultivated with human eyelid tissue derived feeder cells,in which the CFE was 2.2% and the epithelial cell markers P63 and β1-integrin were highly expressed.Conclusion:Human eyelid tissue derived fibroblast cells can be used as feeder layers for expanding engineered human-OECS,providing a new kind of human feeder cells for clinical application.

关 键 词：人口腔黏膜上皮细胞 成纤维细胞 饲养层细胞 眼睑组织 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]