The study of an artificial esophagus constructed of cultured human esophageal wall cells with angiogenetic effects

我们一直在开发人工食道，它是由通过细胞培养方法和细胞外基质增殖的自体细胞构建的。在Sato和Miki之前的一项研究中，我们已经建立了食道上皮细胞和成纤维细胞的共培养方法。我们试图建立一种由人食道上皮细胞、真皮成纤维细胞、主动脉中膜平滑肌细胞组成的人工食管壁，以替代食管壁的弹性和支撑力。将15X105细胞包埋在I型胶原1.5ml6X105细胞中，然后将人成纤维细胞包埋在上述胶原层上的I型胶原0.7ml中。最后，将人食道上皮细胞培养在成纤维细胞的胶原层上。将构建的胶原片体外培养一周，然后移植到无菌大鼠的背阔肌上。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(抗肌动蛋白抗体)。1周后，上皮细胞分层，向胸壁弯曲方向排列密度较低的下层平滑肌细胞与上层成纤维细胞明显不同。下层可见血管生成。两周后，在显微镜下，胶原层似乎重建了上皮层、粘膜下层和适当的肌层。此外，我们还成功地从小鼠的结肠中分离出了肠平滑肌细胞。如果手术切除的标本上皮细胞、成纤维细胞和平滑肌细胞组成的胶原膜能够转化为管腔结构，则人工食道有可能在临床上应用。

We have been developing artificial esophagus which is constructed of autologous cells proliferated by the cell culture methods and extracellular matrix. In a previous study by Sato and Miki, our co-culture methods of esophageal epithelial cells and fibroblasts had been established1. We tried to consturct an artificial csophagus which consisted of human csophageal epithelial cells, dermal fibroblasts, smooth muscle cells isolated from aortic media with the aim of substituting the elasticity and the support of the csophageal wall. 15X105 cells of smooth muscle cells were embedded in 1.5 ml of type I collagen 6X105 cells of human fibroblasts were then embedded in 0.7 ml of type I collagen on the above-described collagen layer with smooth muscle cells. Finally, human esophagcal epithelial cells were cultured on the collagen layer of the fibroblasts. The constructed collagen sheets were cultured in vitro for one week, then transplanted on the lattisimus dorsi muscle of athymic rats. The sheets were examined histologically at one and two weeks later under hematoxillin-eosin stain and immunologicaly stained methods (anti actin antibody). One week later, epithelial cells stratified and the lower layer of smooth muscle cells which arrayed in a low cell density toward the direction of curvature of the chest wall were distinguishable from the upper layer of fibroblasts. And the angiogencsis was recognized in the lower layer. Two weeks later, the collagen sheet seemed microscopically as if the epithelial layer, the submucosal layer and the proper muscle layer were reconstructed.Additionaly, we succeeded in isolating of the intestinal smooth musclecells from the colon of a mice. If the collagen sheet consisted of esophageal epithelial cells, fibroblasts and smooth muscle cells from the surgically resected specimen can be transformed to the luminal structure, possible clinical application of the artificial esophagus is suggested.