Wnt/β－catenin信号调控EMT水平对小鼠胚胎干细胞向肝脏组织结构分化的影响

Effect of Wnt/β-catenin signal regulating EMT level on the differentiation of mouse ESC to liver tissue

目的在前期体外诱导小鼠胚胎干细胞（ESC）向肝系细胞分化基础上，从细胞上皮间质转化（EMT）角度研究ESC分化为肝脏组织结构的可行性。方法选用Balb/c小鼠ESC，首先利用肝细胞生长因子（HGF）、成纤维细胞生长因子（FGF）等体外诱导ESC向肝系细胞方向分化，然后在分化中期（13 d）、成熟期（17 d）两个时间点动态抑制细胞Wnt/β－catenin信号以降低其EMT水平。最后在分化系统中观察分化细胞保持三维组织结构生长情况，检测其中肝系细胞和血管标志物白蛋白（ALB）、血管内皮生长因子（VEGFR）等表达情况，综合判断有无肝脏组织形成，并研究该过程中EMT水平变化的规律和机制。结果ESC分化过程中，分化前期实验组EMT水平与对照组差别不明显，分化中、后期实验组EMT水平较对照组明显降低，分化第18天和20天，实验组上皮钙黏蛋白（E－cad）基因mRNA相对表达水平分别为0.61±0.15和0.47±0.05，对照组分别为0.07±0.05和0；实验组的ALB/AFP/CK8/CK19表达水平在同期内较对照组普遍偏高，标志物CD31/VEGFR1在分化中后期较对照组下降缓慢。在ESC培养上清液中，实验组ALB于第11天可检测到，浓度为（0.32±0.02）mg/L，而对照组ALB为（0.19±0.05）mg/L；实验组尿素于第13天开始检测到，浓度为（8.7±1.0）μmol/L，对照组尿素浓度为（3.1±1.2）μmol/L。ALB和尿素合成在ESC分化系统培养上清中浓度随分化时间延长而明显升高，并且实验组的ALB、尿素浓度值在相同的时间段内明显多于对照组。另外，实验组分化细胞可保持三维组织状态生长，对照组分化细胞最终呈单细胞状态。实验组可检测到肝系细胞标志物以及血管细胞标志物的表达，免疫荧光结果显示类肝细胞和血管结构紧密层状排列，HE染色提示有类肝小叶结构形成，而对照组无血管成分标志物表达。结论ESC向肝系细胞分化中，在分化中、后期降低EMT水平，可有效促进ESC向肝脏组织结构分化。

ObjectiveTo study embryonic stem cell （ESC） differentiation into liver tissue structure from the perspective of epithelial mesenchymal transition （EMT）.MethodsESC of Balb/c mice was selected to induced into hepatic cell using hepatocyte growth factor （HGF）, fibroblast growth factor （FGF） in vitro, and at the time points of metaphase （13 d） and maturity （17 d） of differentiation, dynamic inhibition of Wnt/β-catenin signal was made to reduce the level of EMT. Finally, three-dimensional organization structure growth of the differentiation cells was observed in the differentiation system.Expressions of the liver cells vascular markers[albumin （Alb） and vascular endothelial growth factor （VEGFR）]were detected.ResultsDuring the differentiation of ESC, the level of early EMT in the experimental group and the control group was not significantly different. The level of mid-late EMT in the experiment group was significantly lower than the control group. On the day 18 and 20 of differentiation, the relative mRNA expression level of E-cadherin was 0.61±0.15 and 0.47±0.05 in the experimental group, and 0.07±0.05 and 0 in the control group, respectively.The expression level of ALB/AFP/CK8/CK19 in the experimental group was generally higher than that of the control group in the same period, while CD31/VEGFR1 markers in the experimental group decreased more slowly in the late period of differentiation compared with the control group. In the supernatant of ESC culture, the Alb of the experimental group could be detected onday 7, and the concentration was （0.32±0.02） mg/L, while Alb in the control group was （0.19±0.05） mg/L. Urea in the experimental group could be detected on the day 13, and the concentration was （8.7 ±1.0） μmol/L, and the urea concentration of the control group was （3.1±1.2） μmol/L. The concentration of Alb and urea in the culture supernatant of ESC differentiation system increased significantly with the prolongation of the differentiation time, and the Alb and urea concentrations in the experimental group were significantly higher than those in the control group at the same time period. In addition, the differentiated cells in the experimental group could maintain the growth of three-dimensional tissue, while the differentiated cells in the control group eventually showed a single cell state. The expression of hepatic and vascular cell markers could be detected in the experimental group. Immunofluorescence results showed that the hepatocytes and vascular structures were tightly arranged. HE staining showed the formation of hepatic lobular structure, while the control group had no vascular component markers.ConclusionThe differentiation of ESC into liver tissue can be effectively promoted by decreasing the level of EMT at the mid-late stage of ESC differentiation.