低浓度血清促进胚胎肝祖细胞诱导分化

Promotion in Induction of Differentiation of Hepatic Progenitor Cells by Serum of Low Concentration

为了探讨不同血清浓度诱导培养条件下对体外诱导胚胎肝祖细胞成熟分化的影响。本研究选用含有10%FBS、5%FBS、2%FBS的DMEM/HGF/FGF4肝细胞培养基体外诱导胚胎肝祖细胞的成熟分化,分别于诱导后0d、3d、6d、9d、12dALB-GLuc检测细胞白蛋白的合成水平,细胞计数检测细胞的成长曲线。RT-PCR检测肝细胞相关标志DLK、AFP、CK18,免疫荧光检测ALB、UGT1A的表达情况。ICG摄取和尿素氮检测肝细胞成熟功能。诱导后第三天,ALB-GLuc开始增高,于第9天达高峰,2%FBS组ALB-GLuc读数最高。生长曲线显示低血清浓度下细胞的增殖速度明显慢于高血清浓度。RT-PCR和免疫荧光结果显示诱导后第9天,3个诱导组DLK、AFP表达低于无诱导组,CK18、ALB、UGT1A均高于无诱导组,2%FBS组差异最显著。3个诱导组的肝细胞ICG摄取能力明显增高,2%FBS组ICG阳性细胞数为（60.2±9.0）%,明显高于5%FBS（45.0±3.6）%及10%FBS组（35.2±2.9）%,诱导后肝细胞的尿素合成功能增强,尤其是2%FBS组。低浓度血清培养条件能更好的诱导胚胎肝祖细胞的体外成熟分化。

The aim of the study was to investigate the effect of different serum concentrations on differentiation of embryonic liver progenitor cells induced in vitro. In this study, we used to choose the different concentrations DMEM/HGF/FGF4 induction medium： 10% FBS, 5% FBS, and 2% FBS to induce embryonic hepatic progenitor cells. ALB-GLuc assay was performed to measure ALB synthesis after being induced for 0 d, 3 d, 6 d, 9 d and 12 d, respectively. Cell count was carried out to calculate growth curve. RT-PCR was used to detect hepatic related markers DLK, AFP, CK 18, while immunofluorescence was used to detect the expression of ALB, UGT 1A. ICG uptake and urea nitrogen detection were carried out to detect the function of mature liver cells. 3 days after induction, ALB-GLuc began to increase and arrived to peak at 9 days after induction, 2% FBS induced group had the highest ALB-GLuc activity. The growth curve showed that cell proliferation rate of low serum concentration group was significantly slower than that of high serum concentration group. The results of RT-PCR and immunofluorescence showed that at 9 days of induction, the expression of DLK and AFP of three induction groups was significantly lower than that of control group, whereas the expression of CKI 8, ALB,UGT IA was significantly higher than that of control group, especially the 2% FBS group had the most significant difference. The ICG uptake capacity of three induction groups increased significantly, and ICG positive cells in 2% FBS group was （60.2±9.0）%, significantly higher than （45.0±3.6）% in 5% FBS group and （35.2±2.9）% in 10% FBS group. Urea synthesis function of liver cells after induction enhanced, especially 2%FBS group. Therefore, low concentration of serum culture conditions could better induce embryonic liver progenitor cells in vitro maturation and differentiation.