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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,r EGF)、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。
OBJECTIVE: To establish a primary culture model of rat salivary gland epithelial cells in vitro and to provide seed cells for the study of salivary gland diseases in vitro. Methods: The parotid glands of Wistar rats were removed under sterile conditions for 1 to 2 days. The glands of the Wistar rats were removed under the microscope. The kerotinocyte-SFM was used as the culture medium and the rats were given epidermal growth factor epidermal growth factor (r EGF), bovine pituitary extract (BPE), hydrocortisone (HC), transferrin (Tf) and insulin (INS) Block culture method for culture. Inverted phase contrast microscope was used to observe the morphological characteristics of cultured cells in vitro growth. H-E staining and cytokeratin, vimentin immunohistochemical staining of cultured cells morphological examination and identification. Results: The cultured parotid gland epithelial cells were triangular, polygonal, round, short fusiform, cell monolayer growth, loose connection. H-E staining cells were mostly round, nuclear blue dye, cell protrusions, cell bridges. Positive cytokeratin staining confirmed that cultured cells were epithelial origin; Vimentin, actin and calponin staining cells were mostly positive, to further determine the cells mainly myoepithelial cells. Primary cells maintained good growth within 3 to 5 days and survived for about 1 week. CONCLUSION: Tissue mass culture can be used to obtain rat parotid gland epithelial cells in a simple and rapid manner and establish an in vitro model of parotid gland epithelial cells in Wistar rats.