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

1.我们利用成纤维细胞促进人工食管壁上皮细胞的增殖和分化。方法：将食管成纤维细胞包埋于直径35mm的培养皿中，用聚乙醇酸网状物包埋于I型胶原蛋白溶液中。将食管上皮细胞接种于胶原膜上，用KGM培养。当培养细胞达到融合时，缝合PGA网的两端，制成粘膜管。切开胸大鼠背部，制作背阔肌肌瓣。用纤维胶将粘膜管包裹在肌瓣中。移植后第14天取出移植的粘膜管，检查上皮细胞分层。结果：第14天切除的管腔结构无狭窄，有20层分层。从基底细胞到最上层有大量扁平上皮细胞，呈递增的梯度。上皮细胞分化，离子增多。在胶原层中观察到肌肉层的血管生成。我们通过将平滑肌细胞包埋在成纤维细胞胶原层外，观察平滑肌细胞是否可以作为肌肉层包裹在人工食管壁周围。方法：将人主动脉平滑肌细胞包埋于I型胶原中。然后将来自真皮层的人成纤维细胞包埋在与平滑肌细胞一起的上述胶原层上的I型胶原中。最后，在成纤维细胞的胶原层上培养人食管上皮细胞。将构建的胶原片移植于裸鼠背阔肌上。1周和2周后用苏木希林-伊红染色法和免疫染色法（抗人肌动蛋白抗体：HHF 35）进行组织学检查。结果：1周后提取的胶原片组织学特征如下：平滑肌细胞层位于下层，与成纤维细胞层有明显区别。平滑肌细胞呈低密度排列，呈胸壁弯曲方向排列。上皮细胞分层6-8层。2周后所取薄片的组织学特征如下：1。平滑肌细胞以较高的细胞密度排列在一个曲率方向上。平滑肌细胞和成纤维细胞两层均有血管生成。上皮细胞分层10-13层。上皮层、黏膜下组织层和肌层似乎得到了重建。少

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