摘要
目的:探讨淤胆血清"病理微环境"培养体系诱导胚胎干细胞(ESC)向肝细胞分化的效果及对肝干细胞的筛选作用。方法:将小鼠ESC细胞系E14在无白血病抑制因子培养基中培养,使其自发分化为拟胚体,加入FGF-4和HGF初步诱导,然后置于5%淤胆鼠血清"病理微环境"筛选培养液中继续培养2周,然后进行细胞形态学观察,ALB和CK8/18免疫荧光染色,电镜检查和吲哚氰绿(ICG)摄取试验。结果:经初步诱导分化的ESC置于5%淤胆血清"病理微环境"筛选培养液中培养,初期细胞生长受抑制,部分细胞坏死、凋亡并脱落。1周左右可见上皮样细胞集落呈优势生长。2周左右可见较多肝细胞样集落形成单位(H-CFU),大部分细胞分化为多角形的肝细胞样细胞,呈现较好的均质性,从中央到周边呈逐渐分化成熟的趋势;免疫荧光染色显示ALB和CK8/18表达;电镜检查可见与肝细胞相似的超微结构;吲哚氰绿(ICG)摄取试验可见大量ICG阳性细胞,提示这些细胞具备部分肝细胞特性,且筛选诱导组ICG阳性率显著高于对照组(P<0.05)。结论:采用含淤胆血清的"病理微环境"培养体系从经FGF-4和HGF初步诱导的胚胎干细胞中有效筛选出了具有功能的肝细胞样细胞。
Objective To investigate the effects of pathological microenvironmental culture system consisting of cholestatic sera on the induction of embryonic stem cells （ESC） to differentiate into hepaocyte-like cells and to select them from differentiating embryonic stem cells. Methods El4 cell lines of ESC of mice were cultured in the medium without leukemia suppressing factors to form embryo analogous spontaneously, then were primarily induced by FGF-4 and HGF, and then were added to pathological microenvironmental culture system consisting of 5% cholestatic sera and cultured for at least 2 weeks. Morphological examination, immunofluorescent staining of albumin,and CK8/18 were carried out,and ultrastructure of the cells were observed by electron microscopy. Futhermore, the uptake test of indocyanine green （ICG） was used to evaluate the function and the positive ratio of cell differentiation. Results The proliferation of ESC was inhibited at the early stage when cultured in pathological microenvironmental culture system consisting of 5% cholestatic sera. But 1 week later, epithelioid cell colonies grew dominantly, and in 2 weeks a large number of hepatocyte-like colony-forming unit （H-CFU） were observed, in which most cells exhibiting hepatocellular morphological characteristics, and there was a trend to mature from central to round area. Immunofluorescence staining found red-fluorescence positive cells, indicating the expression of albumin and CK8/18. An uhrastructural analysis revealed a well-developed system of organelles such as mitochondria, lysosomes, Golgi apparatus, and rough and smooth cndoplasmic reticulum. Indocyanine green （ICG） uptake assay was positive for most of the cells, and the positive ratio in experimental group was significantly higher than that in control group （P 〈 0.05）. Conclusion Pathological microenvironmental culture system consisting of 5% cholestatic sera could not only induce ESC to differentiate into hepaocyte-like cells, but also select hepatic stem cells from differentiating ESC initially induced by FGF-4 and HGF.
出处
《实用医学杂志》
CAS
北大核心
2011年第4期558-561,共4页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(编号:30872700
30471799)
广东省自然科学基金资助项目(编号:07001617)
关键词
胚胎干细胞
肝细胞移植
诱导分化
淤胆血清
Embryonic stem cells
Hepatocyte transplantation
Cell differentiation
Cholestatic sera
