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ENGLISH ABSTRACT
人羊膜间充质干细胞体外构建组织工程角膜上皮层的实验研究
肖盼
陈剑
作者及单位信息
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.11.007
Construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells in vitro
Xiao Pan
Chen Jian
Authors Info & Affiliations
Xiao Pan
Department of Ophthalmology, Changshu No.2 People′s Hospital, Changshu 215500, China
Chen Jian
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.11.007
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摘要

背景目前角膜移植手术是治疗严重角膜病变的主要方法，角膜供体来源的匮乏限制该疗法的应用。组织工程角膜为角膜疾病的治疗开辟了新的途径。

目的探讨人羊膜间充质干细胞(hAMSCs)构建组织工程角膜上皮层的可能性。

方法新鲜羊膜组织剪成6 cm×6 cm大小的组织块后用胰蛋白酶＋EDTA消化液消化，将人羊膜上皮细胞(AECs)刮除干净，然后将羊膜组织剪碎，用胶原酶Ⅱ进行消化，分离原代hAMSCs并进行培养。分离新西兰白兔的角膜基质片后按随机数字表法分为2个组，实验组将培养至第3代的hAMSCs以1×10 5/ml的密度种植于去角膜上皮细胞的兔角膜基质片上进行培养，空白对照组为不种植细胞的空白角膜基质片。待细胞达80%～90%融合时再将兔角膜基质片移至插入式培养皿中进行气液界面诱导分化培养，培养至14 d时将兔角膜基质片在质量分数4%多聚甲醛中固定，制备组织切片，行苏木精－伊红染色，观察兔角膜基质片的形态。采用免疫组织化学染色和免疫荧光技术检测兔角膜基质片上诱导分化的hAMSCs中细胞角蛋白3(CK3)和CK12的表达。

结果实验组hAMSCs种植于去角膜上皮细胞的兔角膜基质片上气液界面培养14 d后可形成3～5层的复层细胞，可见兔角膜基底膜的hAMSCs细胞核呈椭圆形，表层的hAMSCs细胞核呈长梭形，形态与正常角膜上皮层相似；而空白对照组兔角膜基质上无细胞生长。免疫组织化学染色显示兔角膜基底膜上的hAMSCs对CK3、CK12呈阳性反应，为细胞质中棕黄色染色，而阴性对照组未见CK3、CK12染色。免疫荧光检测显示兔角膜基底膜上的hAMSCs细胞质中可见CK3、CK12呈绿色荧光，而阴性对照片细胞质荧光缺失。

结论hAMSCs在兔角膜基质片气液界面上可成功诱导分化为角膜样上皮细胞。

羊膜;间充质干细胞;细胞培养;组织工程;角膜上皮;分化;人
ABSTRACT

BackgroundCorneal transplantation is an effective treatment to severe corneal diseases, but the shortage of cornea donor limits its application.Tissue-engineered cornea is being a new approach to corneal diseases.

ObjectiveThis study was to investigate the possibility of construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells (hAMSCs) in vitro.

MethodsFresh human amniotic membranes were obtained under the approval of Ethic Committee of Affiliated First Hospital of Jinan University and informed consent of maternal women.The 6 cm×6 cm amniotic membrane tissue explant was digested using trypsin+ EDTA, and then the amniotic epithelial cells (AECs) were scraped before putting into collagenase Ⅱ digestion medium to isolate hAMSCs.hAMSCs of passage 3 were cultured to achive 80%-90% confluence, and then the cells were incubated on rabbit deepithelial corneal stroma at a 1×10 5/ml density.The corneal stroma was co-cultured with hAMSCs at an air-liquid interface till 14 days.Rabbit deepithelial corneal stroma with and without hAMSCs (experimental group and control group) were fixed in 4% para formaldehyde, and sections were prepared for histopathological examination.Immunochemistry and immunofluorescence were empoyed to detect the expressions of cytokeratin3 (CK3) and CK12 in hAMSCs.

ResultshAMSCs grew well and formed a stratified epidermal structure resembling native corneal epithelium on rabbit corneal stroma in cultured 14 days in the experimental group, with the oval nucleus at basement and fusiform nucleus on the surface of corneal stroma.There was no cell structure in the control group.Immunochemistry revealed brown staining for CK3, CK12 in cytoplasm of hAMSCs on the rabbits corneal stroma, and the green fluorescence for CK3 and CK12 was also seen in the hAMSCs.However, the response for CK3 and CK12 was absent in the control sections either immunochemistry or immunofluorescence test.

ConclusionshAMSCs can be induced to differentiate into corneal epithelioid cells at an air-liquid interface on the rabbit corneal stroma.

Amnion;Mesenchymal stem cells;Cell culture;Tissue engineering;Epithelium, corneal;Differentiation;Humans
Chen Jian, Email: mocdef.3ab61jnehcrd
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引用本文

肖盼,陈剑. 人羊膜间充质干细胞体外构建组织工程角膜上皮层的实验研究[J]. 中华实验眼科杂志,2015,33(11)：991-995.

DOI:10.3760/cma.j.issn.2095-0160.2015.11.007

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世界每年有150万～200万角膜溃疡和眼外伤致盲的新增病例 [ 1 ]。传统眼表重建手术存在供体来源匮乏和免疫排斥反应等问题，自体角膜缘移植或体外培养自体角膜缘干细胞移植是目前治疗角膜缘干细胞缺乏的主要手段，但其来源有限且会对健眼造成损伤，对于双眼角膜缘广泛损伤者更不可行。组织工程角膜将为角膜疾病的治疗开辟新的途径，而构建组织工程角膜的主要问题之一是种子细胞。角膜组织含有角膜上皮细胞、基质细胞和角膜内皮细胞3种细胞成分，目前组织工程角膜重建主要在于上皮层、基质层和内皮层，其中角膜上皮层的重建是当前研究的热点和难点 [ 2 , 3 ]。成体干细胞是存在于机体内各种组织器官中的具有自我更新和分化能力的专能或多能干细胞 [ 4 ]，包括角膜缘干细胞、口腔黏膜上皮细胞、皮肤干细胞、造血干细胞、间充质干细胞等。人羊膜间充质干细胞(human amniotic mesenchymal stem cells，hAMSCs)来源于人胎盘组织的羊膜，具有来源丰富、含量高、免疫原性低、取材方便、无创并且不受伦理学限制等特点，可用于探讨利用角膜基质作为载体将hAMSCs体外诱导分化为角膜样上皮细胞进行眼表重建的可能性。
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