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《Journal of Applied Clinical Pediatrics》 2009-23
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Study on Isolation,Primary Culture and Identification of Murine Extrahepatic Bile Duct Epithelial Cells

CHAI Cheng-wei,ZHENG Shuai-yu,FENG Jie-xiong,WEI Ming-fa,WU Xiao-juan,YANG Ji-xin(Department of Pediatric Surgery,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,Hubei Province,China)  
Objective To develop a duplicable and reliable method for isolation,primary culture of murine extrahepatic bile duct epithelial cells (MEBECs) in vitro.Methods The plastic culture flask was coated by rat-tail collagen typeⅠ.Ten BALB/c mice were given newborn bovine serum (NBS) by intraperitioneal injections for total 7 times injections were sacrificed.Extraheptic bile ducts were disssected and took out,then were minced into 1 mm3 pieces.The pieces were digested with 2.5 g/L trypsin in the presence of 2×104 U/L DNaseⅠand further digested with 2×105 U/L collagenase Ⅳ.MEBECs were isolated and seeded in a culture flask containing DMEM/HamsF12(1:1) supplemented with 100 mL/L fetal bovine serum(FBS) and 10 μg/L epithelial growth factor(EGF).Then the morphologic changes of the cells were observed under an inverted light microscope and the growth curve of the cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide(MTT) assay.The nature of the cells was identified by anti-cytokeratin 19 (anti-CK19)staining and transmission electron microscopy.Results The primary cultured cells attached within 24-48 h of seeding and proliferated in an island-like monolayer.These cells grew vigorously within 1 week and reached the maximum at the 3rd to 4th days and displayed the typical cobblestone appearance.anti-CK19 expressed positive,the cytoplasm showed brown and the cell nucleus was blue.Transmission electron microscope showed typical bile duct epithelia with microvilli on the cells surface,massive mitochondria and rough endoplasmic reticulum in the cells.Conclusions This culture technique might be a reliable method for isolation,purification and primary culture of MEBECs.These cells can serve as a material for advanced study in vitro.
【Fund】: 国家自然科学基金项目资助(30672195);; 湖北省卫生厅重点课题资助(JX1A05)
【CateGory Index】: R575
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