肝细胞生长因子与表皮生长因子联合培养人胆囊上皮细胞的研究

Experimental research of combined hepatocyte growth factor with epidermal growth factor cultured human gallbladder epithelial cell

目的建立体外联合肝细胞生长因子(HGF)和表皮生长因子(EGF)培养人胆囊上皮细胞(HGBECs)方法。方法先剥离上皮,采用反复Ⅳ型胶原酶消化配合刮取HGBECs,制备成单细胞悬液进行接种,细胞分别接种于含或不含10ng/mL EGF以及含10ng/mL HGF+10ng/mL EGF的培养基进行原代培养。倒置显微镜下观察细胞生长的形态变化,细胞计数、噻唑蓝(MTT)法检测HGF+EGF对HGBECs增殖的影响。结果成功培养HGBECs。添加HGF+EGF组较添加EGF组HGBECs增殖速度快且维持时间长;体外存活时间明显延长(19.3±2.5)d vs.(14.2±2.4)d,P<0.05,细胞活力和细胞形态优于只添加EGF组。结论 HGF联合EGF可以明显地促进HGBECs增殖,延长细胞体外存活时间,较长时间稳定细胞形态。

Objective To establish the method of combined hepatocyte growth factor （HGF） with epidermal growth factor （EGF） cultured human gallbladder epithelial cells（HGBECs） in vitro .Methods The epithelial layer was peeled away from human gallbladder ,epithelial layer were digested with collagenase Ⅳ and scraped repeatedly .HGBECs were isolated and seeded in cell cul-ture plates containing medium supplemented with or without 10 ng/mL EGF or with 10 ng/mL HGF and 10 ng/mL EGF respec-tively .Then the morphologic changes of the cells were observed and taken photos with inverted phase contrast microscope ,and counted number of cells ,MTT assay detected vigor of cells in different groups .Results The number of the HGBECs of the HGF＋EGF group was obviously more than the EGF group ,the duration of the HGBECs of the HGF＋ EGF group was obviously longer than the EGF group（19 .3 ± 2 .5）d vs .（14 .2 ± 2 .4）d ,P〈 0 .05 .And the HGBECs of the group with HGF＋ EGF had better cell vigor .Conclusion HGF combines with EGF added to medium can obviously promote the proliferation of HGBECs and prolong the duration and stabilize morphology of HGBECs in vitro .