体外构建犬组织工程化食管的初步试验

背景:近年来组织工程技术的发展为人工食管的研究开辟了新的思路,有研究者应用体外培养的食管黏膜上皮细胞接种于复合高分子材料上,构建组织工程食管获得成功.目的:探讨应用体外培养的犬食管黏膜上皮细胞种植于猪的主动脉脱细胞基质支架上,构建组织工程化人工食管的可行性.设计:观察实验.单位:泰山医学院中心实验室.材料:实验于2004-06/2004-12在泰山医学院中心实验室完成,选用3只出生24 h内杂种犬,由泰山医学院动物园提供. 实验过程中对动物的处置过程符合动物伦理学标准.二氧化碳培养箱MCO-15AC(SANYO),RC-26低温高速离心机(杜邦).胰蛋白酶、转铁蛋白、Ⅱ型胶原酶(GIBCO);DMEM、DispaseⅡ分离酶、鼠抗人角蛋白单克隆抗体(Sigma).方法:用酶-去污剂法对猪主动脉进行脱细胞处理,体外分离、培养、扩增新生杂种犬的食管黏膜上皮细胞,接种于去细胞基质支架体外培养,种植后3天、1周通过组织学及电镜观察食管黏膜上皮细胞在脱细胞基质支架上的生长情况.主要观察指标:体外培养的食管黏膜上皮细胞的形态;脱细胞基质与犬食管上皮细胞的生物相容性.结果:酶化学除垢剂法能使猪主动脉细胞脱落,基质三维结构变疏松.体外扩增的犬食管黏膜上皮细胞可以种植在脱细胞基质上并能生长,增殖.结论:脱细胞的基质与犬食管上皮细胞具有良好的生物相容性,能结合在一起并形成人工食管移植体.

In vitro construction of tissue-engineered esophagus A preliminary test

BACKGROUND:Recently,the development of tissue-engineered technique lms broadened the study of artificial esophagus.Some investigators have inoculated esophageal epithelial cells cultured in vitro onto compound polymer material and successfully constructed tissue-engineered esophagus.OBJECTIVE:To investigate the feasibility of tissue-engineered artificial esophagus by combining dog esophageal epithelail cells and an acelhilarized porcine thoracic aorta allogenic matrix.DESIGN:An experimental observation.SETTING:Central Laboratory,Taishan Medical College.MATERIALS:This study was carried out in the Central Laboratory.Talshan Medical College from June to December in 2004.Three hybrid dogs,24-hour-old,were provided by the Laboratory Animal Center,Taishan Medical College.The protocol was performed in accordance with ethical guidelines for the use and care of animals.The experimenhal instruments and reagents were as follows:CO2 incubator(MCO-15AC,SANYO),hypothermal high-speed centrifuge(RC-26,Dupont),trypsin,transferrin,type Ⅱ collagenase(Gibco),dulbecco's modified eagle's medium(DMEM),DispaseⅡ isolated enzyme,and rat monoclonal anti-keratin antibody(Sigma).METHODS:Acellularization of porcine aortas was performed by a method of enzyme-detergent.Esophageal epithelial cells of hybrid dogs were in vitro isolated,cultured and proliferated.Next,they were inoculated onto an acellularized porcine thoracic aortas allogenic matrix seaffold.Three and seven days later,the growth of esophageal epithelial cells on the acellularized matrix was observed under an electron microscope.MAIN OUTCOME MEASURES:Morphology of esophageal epithelial cells cultured in vitro;Biocompatibility of acellular matrix and dog esophageal epithelial cells.RESULTS:The acellularized procedure resulted in an almost complere removal of the cells and the loose three-dimensional matrix.The acellular matrix could be reseeded with expended esophageal epithelial cells in vitro.and esophageal epithelial cells had the potential of spread and proliferation.CONCLUSION:Acellular matrix possesses satisfactory biocompatibility for allogenic esophageal epithelial cells.Tissue-engineered artificial esophagus can be generated in vitro by a combination of esophageal epithelial cells and allogenic aceilularized matrix.