人皮肤干细胞体外诱导分化构建人工结膜的实验

Transdifferentiation of Skin Stem Cell in Reconstructing Artificial Conjunctiva

目的观察皮肤干细胞在结膜去细胞基质上的生长和分化情况,探讨利用皮肤干细胞及结膜去细胞基质构建人工结膜的可能性。方法将体外分离培养的1～2代人皮肤干细胞接种于人结膜去细胞基质上,在体外观察细胞的分化情况。标本分别行HE染色、人上皮细胞角蛋白CK19单克隆抗体、结膜上皮细胞特异性标记蛋白CK8抗体、CK13抗体、MUC5A/C抗体免疫组化鉴定。结果皮肤干细胞在结膜去细胞基质等模拟的体外微环境下可形成类似结膜上皮细胞的复层增生,并有类结膜上皮细胞特异性标记蛋白表达,呈CK8抗体、CK13抗体、MUC5A/C抗体表达阳性。结论利用皮肤干细胞可在体外模拟的微环境下分化构建人工结膜,用于结膜移植眼表重建。

[Objective] To observe the differentiation and development of skin stem cells on conjunctival extracellular matrix （ECM） and to discuss the possibility of reconstructing artificial conjunctiva with skin stem cells as the seed cells.[Metheds] Pieces of human skin were obtained during operation. And pieces of human conjunctival ECM without epithelium were prepared. The human skin stem cells of the first generation to the second generation were implanted on the human conjunctival ECM. The compound membrane were sliced and underwent immunohistochemistry with human CK8, CK13, MUCSA/C antibody corresponding to the specific surface marker to human conjunctival epithelium, and human dermatic epithelial cell keratin CK19 monoclonal antibody. [Results] Since the 3rd day of culture, the cultured human epithelial cells formed a monohyer, after 7 days formed multiplayer, and histological examination showed intact multiplayer epithelium were human CKS, CK13 antibody and human MUCSA/C antibody positive. [Conclusion] Conjunctival epithelium can be reconstructed from skin stem cell, which may be an alternative for constructing autogenous bioengineered conjunctiva for the clinical therapy of ocular reconstruction.