小分子化合物对小鼠肝细胞分化为胰岛素分泌细胞的体外诱导作用

In vitro differentiation of mouse hepatocytes into insulin-producing cells induced by small molecules

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-q PCR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＇ -deoxycytidine （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 Pdxl and insulin positive cells at both gene and protein levels. Conclusion Small molecules （ 5 - AZA, TSA, RA, ITS, NA） and the sequential 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）.