骨髓间充质干细胞来源类肝细胞的功能和超微结构评估

The evaluation of function and ultramicrostructure for the hepatocyte-like cells derivated from fetal marrow mesenchymal stem cells

目的 对骨髓间充质干细胞 (MMSCs)来源的类肝细胞进行功能和超微结构评估。方法 从人胎儿骨髓中分离MMSCs ,在 1%基质胶 (Matrigel)作基质 ,2 .5 μmol/ml氮胞苷 (AZA)预处理10～ 12h ,肝细胞生长因子 (HGF) 10ng/ml+成纤维细胞生长因子 4 (FGF4 ) 10ng/ml+肝细胞生长培养基 (HGM )中培养和诱导。以未诱导的MMSCs作对照 ,对诱导 4d、7d、14d、2 1d、2 8dMMSCs采用酶联免疫吸附法 (ELISA)检测培养上清液中人白蛋白 (ALB)水平 ,脲酶法检测合成尿素的能力 ,过碘酸希夫试验进行糖原染色 ,电镜观察细胞超微结构。结果 在分化过程的不同时相点检测ALB和尿素生成 ,未诱导分化的MMSCs没有分泌ALB和产生尿素 ,而给予FGF4和HGF处理后 ,MMSCs以时间依赖方式产生ALB和尿素。用过碘酸希夫 (PAS)染色法分析发现MMSCs诱导 14d时首先见到部分细胞出现红紫色染色的糖原积聚物 ,2 8d后阳性染色细胞数量增多 ,提示诱导后MMSCs已具备肝细胞的糖原合成能力。电镜显示未诱导MMSCs具有幼稚细胞的结构特点。诱导 2 8d的MM SCs已分化 ,并具有上皮样细胞的极性结构特点。结论 诱导后MMSCs已具备肝细胞特有的结构和功能性特征。

Objective To evaluate the hepatocyte function and ultramicrostructure of marrow mesenchymal stem cells (MMSCs) derivation hepatocyte like cells. Methods MMSCs were isolated from fetal marrow. MMSCs were cultured and induced in vitro in 1% Matrigel as matrix, 2.5 μmol/ml azacitidine (AZA) pretreatment for 10～12 h, hepatocyte growth factor (HGF) 10 ng/ml+fibroblast growth factor 4 (FGF4) 10 ng/ml+hepatocyte growth medium (HGM). Albumin (ALB) level in culture supernatant was determined with enzyme linked immunosorbent assay (ELISA), capability for dealing with ammonia into urea was detected with urease method, staining for glycogen of undifferentiated MMSCs and MMSCs derivated hepatocyte like cell at induction days 7, 14, 21 and 28 was conducted with periodic acid Schiff's (PAS) test, ultramicrostructure of MMSCs was observed by electromicroscope. Results Undifferentiated MMSCs did not produce ALB and urea. Following treatment with FGF4 and HGF, ALB and urea production by induced MMSCs increased in a time dependent manner. Glycogen storage was first seen by day 14, and maximum levels were seen after day 28, enhancement of glycogen staining for induced MMSCs showed that hepatocyte like cell had glycogen synthesis capability. Undifferentiated MMSCs possessed the construction features of juvenile cell. Induced MMSCs at day 28 had differentiated and had the polarity construction features of epithelioid cell. Conclusions The induced MMSCs has hepatocyte specific construction and functional features.