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

我们一直在研制人工食管，它是由细胞培养方法增殖的自体细胞和细胞外基质构建的。在Sato和三木先前的研究中，我们已经建立了食管上皮细胞和成纤维细胞的共培养方法1。我们尝试用人食管上皮细胞、真皮成纤维细胞、主动脉中膜平滑肌细胞组成人工食管，以替代食管壁的弹性和支撑。将15 × 105个平滑肌细胞包埋在1.5ml I型胶原中，然后将6 × 105个人成纤维细胞包埋在上述具有平滑肌细胞的胶原层上的0.7ml I型胶原中。最后，将人食管上皮细胞培养在成纤维细胞的胶原层上。体外培养1周后，将构建的胶原片移植于无胸腺大鼠背阔肌。分别于术后1周和2周行HE染色和抗肌动蛋白免疫组化染色。1周后，上皮细胞分层，下层平滑肌细胞以低细胞密度排列，朝向胸壁弯曲的方向，与上层成纤维细胞区分开来。血管生成主要发生在下层。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肉层。此外，我们成功地从小鼠结肠中分离出了肠平滑肌细胞。如果由食管上皮细胞、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，则人工食管可能具有临床应用价值。

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.