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目的探索诱导小鼠原始生殖细胞向肝细胞定向分化的最佳条件。方法分离提取小鼠的原始生殖细胞,体外培养扩增后,接种到6孔培养板中。向不同孔中分别加入不同浓度的bFGF、EGF、-βNGF、RA、肝细胞提取液和与胎肝组织分隔培养,进行诱导分化。用靛青绿摄入试验和α-1-抗胰蛋白酶(AAT)、白蛋白(ALB)免疫细胞化学染色鉴定原始生殖细胞向肝细胞分化的状况。结果与胎肝组织分隔培养2 d,可见原始生殖细胞向肝样细胞分化,分化细胞呈圆形和星形,圆形细胞多呈簇状,可以摄入靛青绿呈绿色,并呈AAT和ALB免疫阳性着色,培养12 d时分化率达80%。肝细胞提取液也可诱导原始生殖细胞向肝样细胞分化,12 d时分化率达70%以上;0.5 g/L和1 g/L的肝细胞提取液的诱导分化率无显著差异;但是0.5 g/L提取液诱导组圆形细胞较多;而1 g/L肝提取液诱导组,星状分化细胞多。β-神经生长因子(-βNGF)诱导4 d,可见肝样细胞出现,随诱导时间延长,分化细胞增多,圆形分化细胞较多,12 d时分化率达60%以上;20μg/L和50μg/L两种浓度的-βNGF诱导分化率无显著差异。bFGF、EGF、RA诱导组未见ALB免疫反应阳性细胞。RA可以增强-βNGF的诱导分化作用,使星状细胞增多明显。结论-βNGF和肝细胞因子可诱导原始生殖细胞向肝细胞定向分化。
Objective To explore the optimal conditions for inducing mouse primordial germ cells to differentiate into hepatocytes. Methods The mouse primordial germ cells were isolated and cultured. After in vitro culture, the cells were inoculated into 6-well culture plates. Different concentrations of bFGF, EGF, -βNGF, RA, hepatocyte extract and fetal liver tissue were separately cultured in different wells to induce differentiation. The condition of primary germ cells differentiated into hepatocytes was identified by indocyanine green ingestion test and α-1-antitrypsin (AAT) and albumin (ALB) immunocytochemical staining. RESULTS: The primary germ cells differentiated into hepatocyte-like cells after differentiation for 2 days. The differentiated cells were round and star-shaped. The round cells were mostly clustered, which could ingest green indocyanine green and showed AAT and ALB Immunopositive staining, differentiation rate of 80% cultured 12 d. Hepatocyte extracts also induced differentiation of primary germ cells into hepatocyte-like cells, with a differentiation rate of over 70% at 12 days. There was no significant difference in the induction rates of hepatocyte extracts at 0.5 and 1 g / L There were more round cells in g / L extract group, while 1 g / L liver extract induced more star-shaped cells. When induced by β-nerve growth factor (β-NGF) for 4 days, hepatic cells appeared. With the prolongation of induction time, the number of differentiated cells increased, and the numbers of round differentiated cells were more than 60% / L two concentrations of-βNGF induced differentiation rate was no significant difference. There was no ALB positive cells in bFGF, EGF and RA groups. RA can enhance the induction of differentiation of-βNGF, so that astrocytosis increased significantly. Conclusion-βNGF and hepatocyte factor can induce the primordial germ cells to differentiate into hepatocytes.