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Development of a Hybrid Artificial Esophagus Using Cultured Human Esophageal Epithelial cells, fibloblasts, smooth muscle cells

使用培养的人食管上皮细胞、成纤维细胞、平滑肌细胞开发混合人工食管

基本信息

批准号：
07671421
负责人：
ANDO Nobutoshi
金额：
$ 1.54万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

1.We utilized fibroblasts to acelerate proliferation and differentiation of epithelial cells of the artificial esophageal wall. Methods : Esophageal fibroblasts were embedded in type I collagen solution with polyglycolic acid mesh in a 35mm diameter culture dish. Esophageal epithelial cells were seeded on the collagen sheets and cultured with KGM.When the culture cells reached confluency, both edges of the PGA mesh were sutured to make a mucosal tube. The backs of athymic rats were incised and muscle flaps in the latissimus dorsi muscle were made. The mucosal tubes were wrapped in the muscle flaps using fibrine glue. Grafted mucosal tubes were removed on the 14th day after transplantation and epithelial cell stratification examined. Results : Luminal structures removed on the 14th day also did not have stenosis, and had twenty layr stratification. Numerous flat epithelial cells in an increasing gradient from the basal cells to the uppermost layr were found. Epithelial cell differentiat … More ion was also observed. Angiogenesis from the muscle layr was observed in the collagen layr.2.We examined whether smooth muscle cells could be surrounded around the artificial esophageal wall as a muscle layr by laminificating collagen sheet embedding smooth muscle cells outside of the collagen layr of fibroblasts. Methods : Human aortic smooth muscle cells were embedded in type I collagen. Human fibroblasts from dermis were then embedded in type I collagen on the abovementioned collagen layr with smooth muscle cells. Finally, human esophageal epithelial cells were cultured on the collagen layr of the fibroblasts. The constructed collagen sheet were transplantated on the lattisimus dorsi muscles of nude rats. The sheets were examined histologically at one and two weeks later under hematoxillin-eosin stain and immunological stain methods (anti human actin antibody : HHF 35). Results : The histological features of the collagen sheet extirpated one week later were as follows : 1.The layr of smooth muscle cells was found in the lower layr and was clearly distinguishable from the layr of fibroblasts, 2.Smooth muscle cells arrayd layr to layr in a low cell density in a direction of curvature of the chest wall, 3.Epithelial cells stratified 6-8 layrs. The histological features of the sheet extirpated two weeks later were as follows : 1.Smooth muscle cells arrayd in a high cell density in a direction of the curvature, 2.Angiogenesis was found in both layrs of the smooth muscle cells and fibroblasts, 3.Epithelial cells stratified 10-13layrs. It seemed as if the epithelial layr, submucosal tissue layr and proper muscle layr were reconstructed. Less

1.利用成纤维细胞加速人工食管壁上皮细胞的增殖和分化。方法：在直径35 mm的培养皿中，将食道成纤维细胞包埋于含有聚乙醇酸网状物的I型胶原溶液中。将培养的食道上皮细胞接种于胶原片上，与KGM共同培养，待细胞融合后，缝合PGA网两边，形成粘膜管。切开大鼠背部，制作背阔肌肌皮瓣。黏膜管用纤维素胶包裹在肌瓣中。移植后第14天取出移植物黏膜管，行上皮细胞层层检查。结果：第14天摘除的管腔结构也未见狭窄，有20层复层。可见大量扁平上皮细胞，从基底层到最上层呈递增趋势。…上皮细胞分化观察到较多的离子。2.通过在成纤维细胞的胶原层外包埋肌层细胞的胶原片，观察在人工食管壁周围是否可以将平滑肌细胞作为肌层包裹。方法：将人主动脉平滑肌细胞包埋于I型胶原中。然后将真皮成纤维细胞包埋于上述胶原层上的I型胶原中，并与平滑肌细胞一起。最后，将人食道上皮细胞培养在成纤维细胞的胶原层上。将构建的胶原膜移植到裸鼠背阔肌上。分别于术后1周和2周进行组织学检查，苏木精-伊红染色和免疫组织化学染色(抗人肌动蛋白抗体：HHF-35)。结果：1周后摘除的胶原膜的组织学特征为：1.下层可见平滑肌细胞层，与成纤维细胞层明显不同；2.光滑的肌细胞沿胸壁弯曲方向排列成低密度层；3.上皮细胞层6~8层。2周后摘除标本的组织学特征为：1.肌细胞呈高密度排列，细胞密度高；2.成纤维细胞和平滑肌细胞均有血管生成；3.上皮细胞层数为10-13层。上皮层、粘膜下组织层和固有肌层似乎得到重建。较少