摘要
目的建立一种专门用于小鼠肝脏前体细胞的培养条件,并鉴定该条件在连续传代培养的过程中是否能够维持前体细胞良好的生长状态,并减少分化的发生。方法配制4种不同配方的培养基,在连续传代过程中通过观察细胞形态学变化而选择适用的配方,并经Western blot检测细胞内肝细胞标志物白蛋白(albumin,ALB)、肝脏前体细胞标志物甲胎蛋白(α-fetoprotein,AFP)和胆管细胞标志物细胞角蛋白-19(cytokeratin-19,CK-19)表达量的变化,流式细胞术分析细胞周期,以及免疫荧光染色鉴定前体细胞标志物的表达以进一步验证选择配方的有效性。结果使用第4种培养基进行培养时,细胞形状规则,边界清晰,生长活力较好,连续传代培养后细胞的形态未发生明显改变。Western blot检测显示,ALB、AFP和CK-19在P0代、P3代和P8代中的表达量没有显著改变。流式细胞术结果显示,经连续多次传代的细胞约76%的细胞处于G0/G1期,近24%的细胞处于S+G2/M期,而且约89%的细胞表达肝脏前体细胞标志物上皮细胞粘附分子(epithelial cell adhesion molecule,EpCAM)。免疫荧光染色进一步证实了大多数细胞表达EpCAM。结论建立了合适的培养基配方可用于小鼠成体肝脏前体细胞的连续传代培养,为更加深入地研究肝脏前体细胞的机制与再生医学方面创造了前提条件。
Objective To establish a specific formula of culture condition for mouse hepatic progenitor cells and identify wheth-er the cells can be maintained in good condition and kept from differentiation during serial passage under this condition. Methods Four different types of media were prepared. The optimal medium was determined based on the morphological changes during serial passage. Expression levels of hepatic marker albumin （ALB）, hepatic progenitor cell marker alpha-fetoprotein （AFP）, and bile duct epithelium marker cytokeratin-19 （CK-19） were detected by Western blot. The cell cycle was analyzed by flow cytometry, and the expression of progenitor cell markers was verified by immunofluorescent staining to further assess the effectiveness of the selected culture medium. Results Cells in the 4th culture medium grew energetically with regular shapes and clear boundaries, and no obvious change in mor-phology was observed after serial passage. Western blot revealed that ALB, AFP, and CK-19 were expressed stably in P0, P3 and P8 cells. Flow cytometry results showed that after serial passage, about 76% of the cells were in G0/G1 stage and approximately 24% of the cells were in S ＋ G2/M stage, while 89% of the cells expressed epithelial cell adhesion molecule （EpCAM）, a typical hepatic pro-genitor cell marker. The expression of EpCAM was further confirmed by immunofluorescent staining. Conclusion A specific formula of medium for serial passage of adult mouse hepatic progenitor cells has been established, which provides preconditions for further study of the mechanism of hepatic progenitor cells growth and regenerative medicine.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2016年第5期447-453,共7页
Chinese Journal of Histochemistry and Cytochemistry
基金
国家自然科学基金(31371169)
关键词
小鼠成体肝脏前体细胞
连续传代
可塑性
培养条件
Adult mouse hepatic progenitor cells
serial passage
plasticity
culture condition
