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目的 探讨体外快速培养犬口腔黏膜上皮细胞(OMECs)及其与猪小肠黏膜下层(SIS)体外复合培养的方法,为组织工程化黏膜研究提供实验依据.方法 采用混合酶消化法,于6%胎牛血清的上皮细胞无血清培养基(DKSFM)中培养OMECs,观察OMECs的形态特征、绘制生长曲线并进行细胞表面标志物检测.将第2代犬OMECs接种在SIS上,行苏木精一伊红染色、免疫组织化学染色、扫描电镜等观察OMECs在SIS上的生长状况.结果 OMECs在DKSFM中生长良好,CK19细胞角蛋白免疫组化染色阳性.OMECs接种在SIS上呈单层生长,多角形,铺路石样排列,复合培养8 d呈多层生长.结论 采用混合酶消化法,用含6%胎牛血清的DKSFM培养犬OMECs,方法简便,适合推广应用.SIS与OMECs有良好的相容性,可作为构建组织工程化黏膜的理想支架材料.“,”Objective To explore an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering. Methods The primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immuno histochemical staining, and scanning electron microscope (SEM). Results OMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone. Conclusion Culture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.