体外培养人胆囊上皮细胞的形态学及培养条件的优化

The Morphological Characteristics and Optimized Culture Condit ion of Human Gallbladder Epithelial Cells in Vitro

目的 探索人胆囊上皮细胞的最佳分离方法和适宜的体外生长条件 ,为深入研究胆囊的生理功能和相关疾病的病理机理奠定基础。方法 用Ⅳ型胶原酶消化及钝性刮离法分离胆囊上皮细胞 ,两步贴壁法纯化。比较层粘连蛋白、多聚赖氨酸、纤维连接蛋白等不同培养基质对传代细胞生长的影响。光镜及电镜下观察细胞的形态和超微结构。结果 每个胆囊可分离得到 ( 1～ 5)× 10 7个胆囊上皮细胞 ,细胞活性可达 90 %。细胞呈典型的扁平多角状、柱状形态 ,片状贴壁生长 ,上皮细胞特异性抗原CK19表达阳性。结论 Ⅳ型胶原酶消化法加钝性刮剥分离法和两步贴壁法可成功分离高活性、高纯度的人胆囊上皮细胞 ;纤维连接蛋白包被的培养器皿和含 2 0 %小牛血清及

Objective To explore the suitable met hod for isolation and maintenance of primary cultures of human gallbladder epith elial cells (GECs) for establishing the basis of research works in physiological function of gallbladder and its related diseases.Methods GECs were isolated with collagenase typ e Ⅳ and blunt separation.The dishes were coated with fibronectin, laminin and p oly-D-lysine respectively.Additional 10 ng/ml epidermal growth factor was adde d to DMEM medium containing 20% fetal calf serum.The cells were studied under light and electron microscope to determine their shape and distrib ution.Results Each gallbladder yielded approximately (1-5)×10 7columnar epithelial cells,greater than 95% of which were viable by trypan blue exclusion.The cells grew vigorously within one week w hich was flat and multangular in shape. CK19 expressed positive.Electron microsc ope showed typical gallbladder epithelia with microvilli ,tight junctions and mucus droplets.Conclusion Combination of collagenase type Ⅳ, mechanical blunt separation and two-step attachment is of great benefit for sep arating and harvesting GEC.Fibronectin coated culture dish and DMEM medium conta ining 20% calf serum and 10 ng/ml hEGF is of great benefit for c ulturing gallbladder epithelial cells.