显微剥离酶消化法体外培养人胚胎食管鳞状上皮细胞的方法研究

HUMAN EMBRYONIC ESOPHAGUS SQUAMOUS EPITHELIAL CELLS CULTURED THROUGH MICROSCOPIC STRIPPING TECHNIQUE AND DIGESTION IN VITRO

目的探讨利用显微剥离酶消化法行人胚胎食管鳞状上皮细胞原代培养的可行性,并对所得细胞进行纯化及传代培养。方法选择意外流产20周龄胎儿食管标本,通过显微剥离法获得菲薄食管黏膜上皮,经短时酶消化分离所得细胞,在10%胎牛血清混合培养基中培养,通过倒置显微镜、透射电镜及免疫组织化学方法鉴定所得细胞,并测定其生长曲线。结果倒置显微镜下见细胞呈不规则圆形或多边形,透射电镜可见其具有鳞状上皮细胞特征性的张力微丝,细胞表面微突起,细胞角蛋白免疫组织化学染色胞浆呈棕黄色阳性表现,MTT法测定第2代细胞在传代培养3~4d达生长高峰。3、4d吸光度(A)值与1、2、5及6d比较,差异有统计学意义(P<0.05)。结论通过显微剥离酶消化法可获得足量、较纯食管鳞状上皮细胞,混合培养基中细胞能快速传代、扩增,适宜作为种子细胞构建组织工程食管。

Objective To discuss the applycation possibility of the microscopic stripping technique used in the primary culture of human embryonic esophagus squamous epithelial cells, and of the methodds for the isolation, depuration and subculture of the esophagus epithelial cells in vitro. Methods The squamous epithelial cells wereobtained from the esophagus mucous membrane of the 20-week abortion fetus through the microscopic stripping technique, and were digested with trypsin. Then, the morphological, immunohistochemical observation and the growth curve of the isolated cells were studied. Results The isolated cells were spherical in the cell suspension and spherical- like or polygon-like after attachment to the culture flask. The squamous epithelial specialized cytokeratin staining was strongly positive. And the morphological studies by the transmission electron microscopy indicated that the cultured cells were squamous epithelial cells. The squamous epithelial cells reached the peak level 3-4 days after the transfer of the culture. The absorbance at 3 and 4 days was significantly higher than that at 1,2,5 and 6 days （P^0.05）. Conclusion A large mumber of squamous epithelial cells can be available with the microscopic stripping technique and the digestion method. The cultured squamous epithelial cells can be proliferated quickly, and fit for the tissue engineering study.