目的：分离原代小鼠肝细胞，在小分子化合物作用下将其诱导分化为胰岛素分泌细胞（ IPCs ）。方法采用肝脏组织直接剪碎的方法分离、培养小鼠肝细胞，并进行肝细胞糖原染色鉴定。随后将肝细胞分别用不同的诱导剂5－氮杂胞苷（5－AZA）、曲古霉素A（TSA）、维甲酸（RA）、胰岛素－转铁蛋白－硒（ITS）、尼克酰胺（ NA ）及不同糖浓度的培养液进行诱导，同时以不含诱导剂的培养液培养肝细胞作为阴性对照。结果原代肝细胞在5－AZA、TSA的作用下去分化，RT－qPCR显示成熟肝细胞表面标志物白蛋白明显下降，出现巢蛋白（ nestin）表达阳性细胞，进一步在含RA、ITS的低糖培养液培养7 d及含NA的低糖培养液培养7 d，分别获得在基因及蛋白水平都表达的Pdx1及胰岛素阳性细胞。结论小分子化合物（5－AZA、TSA、RA、ITS、NA）及依次使用高浓度和低浓度葡萄糖可以在短时期内（17 d）将肝细胞诱导为胰腺前体细胞（Pdx1阳性细胞），并进一步分化为IPCs。%Objective To induce differentiation of isolated primary mouse hepatocytes into insulin-producing cells ( IPCs) with small molecules.Methods Mouse hepatocytes were isolated and cultured from minced mice livers be-fore being identified by glycogen staining.They were subsequently treated with the following inducers, 5-aza-2′-de-oxycytidine (5-AZA), richostatin A (TSA), retinoic acid (RA), insulin-transferrin-selenium (ITS), nicotinamide ( NA) and with culture media of different glucose concentration.Hepatocytes cultured without inducers were served as the negative control.Results Primary hepatocytes underwent dedifferentiation in the effect of 5-AZA and TSA.RT-qPCR demonstrated significant decrease of albumin, a surface marker of mature hepatocytes, along with nestin-positive cells. Further culture with low glucose medium containing either RA plus ITS or NA alone for 7 d yielded Pdx1 and insulin posi-tive cells at both gene and protein levels.Conclusion Small molecules (5-AZA, TSA, RA, ITS, NA) and the se-quential use of high and low concentration of glucose can induce hepatocytes into pancreatic precursor cells, and further differentiate into insulin-producing cells in a short period (17 d) .
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