中文摘要：

目的比较三维条件下不同代次角质形成细胞构建的表皮形态及增殖分化情况。方法采用不同代次角质形成细胞构建组织工程皮肤，通过苏木精-伊红（HE）和高碘酸-希夫（PAS）染色，观察各组组织工程皮肤的结构形态；并利用角蛋白CK1／CK10、CK5／CK14、细胞增殖核抗原Ki-67免疫组织化学染色，比较各组组织工程皮肤表皮的增殖分化能力。结果不同代次角质形成细胞构建的组织工程皮肤都有明显的表皮真皮结构。1、2代较其他代次分层情况好，2代表皮层厚度明显高于其他代次（P〈0．05）。加Ⅳ型胶原组的表皮真皮间黏附的紧密程度高于未加Ⅳ型胶原组，Ⅳ型胶原对表皮的厚度影响不大（P〉0．05）。Ⅳ型胶原组PAS染色阴性，说明单独加Ⅳ型胶原不能形成基底膜结构。2代角质形成细胞Ki-67增殖系数接近正常皮肤，其余各代次角质形成细胞的增殖系数逐渐减少（P〈0．05）。CK1／CK10、CK5／CK14在1、2代皮肤中阳性表达明显，其余代次对于两种角蛋白的表达并不明显。结论3代以前角质形成细胞有更好的增殖分化能力，更适合作为组织工程皮肤的种子细胞。

英文摘要：

Objective To compare the epidermal shape built by different passages of keratinocytes and its ability of proliferation and differentiation in three-dimensional conditions. Methods Different passages of keratinocytes were used to construct tissue-engineered skin. The morphology of the tissue-engineered skin was observed with HE and PAS staining, while CK1/CK10, CK5/CK14, Ki67 were detected by immunohistochemieal assays. Results All the tissue-engineered skin had a significant dermoepidermal structure. The stratification of 1st and 2nd passage skins were better, and 2nd passage epidermis was thicker than that in other passages （P〉0. 05）. Dermoepidermal structure in collagen type IV group binded more tightly, but collagen type Ⅳ had little effect on the thickness of the epidermis （P〉0.05）. In collagen type IV group PAS stain was negative, indicating type IV collagen was unable to promote the reconstruction of BM in vitro. The Ki-67 proliferation index of the 2nd keratinocyte was similar to the normal skin, the remaining passages keratinocyte proliferation gradually decreased （P〉0.05） ; the 1st and 2nd passage skins expressed CKI/CK10 and CK5/CK14. Conclusions Keratinocytes before the 3rd passage have a better ability in the proliferation and differentiation, and so they are more suitable as seed cells for tissue-engineered skin.