食管黏膜上皮细胞与SIS复合培养及生物学特性研究  

AN EXPERIMENTAL STUDY OF COCULTURE OF ESOPHAGEAL MUCOSA EPITHELIAL CELLS WITH SIS AND THEIR BIOLOGICAL CHARACTERISTICS

作　　者：谭波[1] 魏人前[1] 杨志明[1] 李秀群[1] 韩平[1] 智伟[1] 解慧琪[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2008年第6期742-746,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30670734);四川省科技厅青年基金资助项目(04ZQ026-038)~~

摘　　要：目的探讨体外快速培养犬食管黏膜上皮细胞(esophageal mucosa epithelial cells,EMECs)及其与SIS体外复合培养的方法,为组织工程食管研究提供实验依据。方法取2～5周龄幼犬5只,无菌获取其食管组织,采用混合酶消化法,分离培养EMECs,于倒置相差显微镜下观察细胞生长情况,取第2代细胞培养1～5 d绘制生长曲线并进行细胞表面标志物细胞角蛋白19(cytokeratin 19,CK-19)检测。取第2代EMECs与SIS复合培养4、8 d,行形态学、组织学及扫描电镜观察。结果倒置相差显微镜下EMSCs呈典型铺路石样。CK-19免疫组织化学染色可见细胞胞浆呈阳性染色。MTT法测定第2代细胞在传代培养2 d生长达高峰。EMECs与SIS复合培养4 d,细胞呈多角形,细胞间连接紧密,呈铺路石样排列;SIS表面形成1～2层细胞组成的复层上皮样结构。8 d时,细胞密度增大,SIS表面形成2～3层细胞组成的复层上皮样结构;扫描电镜见细胞在材料上已大量增长,呈层状排列。结论采用混合酶消化法,用含6%FBS的DKSFM培养犬EMECs,方法简便,适合推广应用。SIS与EMECs有良好的相容性,可作为构建组织工程食管的理想支架材料。Objective To explore an effective method to cultivate esophageal mucosa epithelial cells （EMECs） of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium （DKSFM） containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker （cytokeratin 19, CK-19） were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged like slabstone. Immunohistochemical staining of CK-19 of the 2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. EMECs were polygon in shape and arranged like slabstone, and formed a single layer on the surface of SIS. The cells were contacted closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, and showed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS shows good biocompatibility and can be used as a good scaffold material in the tissue engineered esophagus.

关 键 词：食管黏膜上皮细胞 SIS 复合培养 生物学特性 犬 

分 类 号：R318.1[医药卫生—生物医学工程] Q813.1[医药卫生—基础医学]