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目的:通过miRNA-122(miR-122)转染胚胎干细胞(ESCs)源性肝前体细胞,观察及评估其是否能推进ESCs向肝细胞的分化过程。方法:采用丁酸钠、成纤维细胞生长因子4(FGF-4)及地塞米松(Dex)联合序贯诱导小鼠ESCs使之初步分化为肝前体细胞,然后利用Gateway技术构建出pAV.Ex1d-CMV>miR122/IRES/eGFP重组腺病毒表达载体,使其转染贴壁诱导第9天的小鼠ESCs源性肝前体细胞,获得稳定高表达miR-122的细胞后继续培养。采用荧光显微镜观察转染后细胞形态的变化;使用real-time RT-PCR检测肝特异性基因的表达;免疫荧光检测肝特异性蛋白表达水平;吲哚氰绿摄取实验、糖原染色及尿素合成功能实验检测肝细胞功能。结果:丁酸钠、FGF-4及Dex联合序贯诱导可将小鼠ESCs成功地诱导为肝前体细胞。转染miR-122后,细胞在形态学上更接近成熟肝细胞;肝特异性基因白蛋白(ALB)、甲状腺素运载蛋白、α1抗胰蛋白酶、葡萄糖-6-磷酸酶、细胞角蛋白8、胆固醇7α-羟化酶及细胞色素P450 3A4的mRNA表达均增高;肝特异性蛋白ALB及细胞角蛋白18均表达增高,而甲胎蛋白表达降低;吲哚氰绿摄取实验、糖原染色及尿素合成功能实验结果均显示转染miR-122后肝细胞功能较对照组明显增强。结论:丁酸钠、FGF-4及Dex联合序贯诱导可将小鼠ESCs成功地诱导为肝前体细胞;miR-122能够有效地促进小鼠肝前体细胞的分化与成熟。
OBJECTIVE: To transfect embryonic stem cells (ESCs) derived liver precursor cells (miSCs) by miRNA-122 (miR-122) and to observe whether they can promote the differentiation of ESCs into hepatocytes. Methods: Mouse ESCs were initially induced to differentiate into hepatic progenitor cells by sodium butyrate, fibroblast growth factor 4 (FGF-4) and dexamethasone (Dex), and pAV was constructed by Gateway technique. Ex1d-CMV> miR122 / IRES / eGFP recombinant adenovirus expression vector was transfected into mouse ESCs-derived hepatic progenitor cells on the 9th day after transfection and transfected into cells stably expressing miR-122. The morphological changes of cells were observed by fluorescence microscopy. Liver-specific gene expression was detected by real-time RT-PCR. Liver-specific protein expression was detected by immunofluorescence. Indocyanine green uptake assay, glycogen staining and urea synthesis Function test to detect liver cell function. Results: The sequential induction of sodium butyrate, FGF-4 and Dex induced mouse ESCs successfully into hepatic precursor cells. Cells transfected with miR-122 were morphologically closer to mature hepatocytes; liver-specific gene albumin (ALB), thyroxine transporter, α1 antitrypsin, glucose-6-phosphatase, cytokeratin 8, Cholesterol 7α-hydroxylase and cytochrome P450 3A4 mRNA expression increased; liver-specific protein ALB and cytokeratin 18 were increased, while the expression of alpha-fetoprotein decreased; indocyanine green uptake experiments, glycogen staining and urea The results of the synthetic functional assays showed that the function of hepatocytes transfected with miR-122 was significantly enhanced compared with the control group. Conclusions: The sequential induction of sodium butyrate, FGF-4 and Dex can successfully induce mouse ESCs into hepatic precursor cells. MiR-122 can effectively promote the differentiation and maturation of mouse hepatic precursor cells.