大鼠唾液腺细胞体外原代培养的一种新方法

目的 建立大鼠唾液腺上皮细胞体外原代培养模型,为体外研究唾液腺疾病提供种子细胞。方法 在无菌条件下取出生1~2 d的Wistar大鼠腮腺组织,手术显微镜下去除腺体包膜,以无血清培养基( kerotinocyte-SFM )为培养液,并添加表皮生长因子( rat epidermal growth factor, rEGF )、牛垂体提取物(bovine pituitary extract,BPE)、氢化可的松(hydrocortisone,HC)、转铁蛋白(transferrin,Tf)、胰岛素(insulin,INS)等因子,应用组织块培养法进行培养。用倒置相差显微镜观察培养细胞体外生长的形态特征。用H-E染色及细胞角蛋白、波形蛋白免疫组织化学染色对培养的细胞进行形态学检查和鉴定。结果 培养的腮腺上皮细胞为三角形、多边形、圆形、短梭形,细胞单层生长,连接疏松。H-E染色可见细胞多为圆形,核蓝染,细胞有突起、细胞间桥。细胞角蛋白染色阳性,证实所培养细胞为上皮来源;Vimentin、actin和calponin染色细胞大部分呈阳性,进一步确定细胞主要为肌上皮细胞。原代细胞在3~5 d内保持良好的生长状态,并存活1周左右。结论 组织块培养法可以简捷、快速地获得大鼠腮腺上皮细胞,成功建立Wistar大鼠腮腺上皮细胞的体外模型。

A new method for primary culture of salivary gland cells of rats in vitro

PURPOSE： To establish the primary culture model of salivary gland epithelial cells from Wistar rats in vitro and provide seeds cells for studies of salivary diseases. METHODS： The parotid gland was obtained from Wistar rats which were 1-2 days old under sterile conditions, and the gland capsule was removed under the operating microscope. The parotid gland cells were cultivated with tissue block method in serum-free medium （kerotinocyte, SFM） containing rat epidermal growth factor （rEGF）, bovine pituitary extract （BPE）, hydrocortisone, transferrin, insulin, etc. The morphology of the culture cells was observed under inverted phase contrast microscope. The cells were identified by H-E staining and cytokeratin, vimentin immunohistostaining. RESULTS： The morphology of parotid gland epithelial cells was triangle, polygon, round or short spindle, and the cells displayed monolayer growth with loose connections. H-E staining showed the cells were round, with blue-stained nuclei, projections and intercellular bridge. The cells were positive for cytokeratin, which confirmed that the cultured ceils were from epithelium origin. Most cells were positive for vimentin, actin and calponin, which further determined the cultured cells were mainly myoepithelial cells. The primary cells grew well in 3-5 days, and could survive for about one week. CONCLUSIONS： The parotid gland epithelial cells of Wistar rat are simply and quickly cultivated with tissue block method. The primary culture model of parotid gland epithelial cells in vitro was successfully established.