摘要
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 investigate the better factor for inducing the primordial germ cells to differentiate into hepatocytes in vitro. Methods The primordial germ ceils （PGCs） from the gonadal ridges of the mouse embryos at 11 days postcoitum （dpc） from Kunming pregnancy mice were cultured and proliferated in vitro. An aliquot PGCs suspension was cultured in 6-well dishes for 24 h. Then bFGF, EGF, β-NGF, RA and hepatocyte abstract were added into the dishes respectively, Anather aliquot PGCs were co-cultured with mouse embryonic hepatocytes for directional differentiation . The morphological changes and indiocyanine green （ICG） intake of the PGCs were observed; albumin （ALB） and α-1-antitrypsin （AAT） were assayed by immunocytochemistry. Results After the addition of β-NGF, hepatocyte abstract and CM-EH the ICG was intaken by the differentiated cells which like star and ovum, the ALB and AAT immune positive expression were detected in those differentiated cells. The ratio of positive cells was above 60% in 2 weeks. But bFGF, EGF and RA had no same as the effects. However RA could improve the ratio of differentiated cells induced by β-NGF and hepatocyte abstract. Conclusion β-NGF and some factors from hepatocytes and embryonic hepatocytes could effectively induce PGCs to differentiate into hepatocytes.
出处
《解剖学报》
CAS
CSCD
北大核心
2005年第6期650-654,共5页
Acta Anatomica Sinica
基金
山东省科技厅攻关计划项目(01BB1DADA1)
