人肝细胞核因子4α联合组蛋白去乙酰化酶和甲基化转移酶抑制剂诱导成纤维细胞转分化为肝细胞的研究  

Experimental study on the induction of human hepatocytes into fibroblasts in vitro by nuclear factor 4α combined with histone deacetylase inhibitor and DNA methyltransferase inhibitor

作　　者：邓洁敏 伍耀豪[1] 曾乐祥[1] 蒋雯丽[1] 张杰[1] 周嘉嘉[1] 邱荣林[1] 陈子月[1] 邓小耿[1] 

机构地区：[1]中山大学孙逸仙纪念医院小儿外科,广州510289 [2]广州中医药大学附属第二医院广东省中医院麻醉科,510120

出　　处：《中华实验外科杂志》2017年第9期1496-1499,共4页Chinese Journal of Experimental Surgery

基　　金：广东省自然科学基金（2015A030313037）;广州市科技计划项目（201604020151）

摘　　要：目的 利用人肝细胞核因子4α（HNF4A）与表观遗传修饰[组蛋白去乙酰化酶抑制剂（HDACi）＋甲基化转移酶抑制剂（DNMTi）]联合诱导人成纤维细胞向肝细胞转分化，建立更为高效、新型的肝细胞转分化方法。 方法取第6～8代人皮肤成纤维细胞，置于5’氮杂胞苷（5-AzaC）与丙戊酸（VPA）中，并感染HNF4A重组慢病毒共同诱导20 d。用倒置相差显微镜动态观察诱导过程中的细胞形态变化，实时定量聚合酶链反应（Real-time PCR）检测并分析肝细胞特异性基因的表达，吲哚菁绿摄取实验、糖原过碘酸-无色品红（PAS）染色和尿素合成功能实验验证生物学功能。 结果 诱导过程中，细胞形态逐渐由长梭形转变为上皮样形态，并逐渐开始过表达肝特异基因及表现生物学功能。诱导第20天，细胞在形态学上有明显肝细胞上皮样变化，胞质丰富、核大而圆深染且核仁明显；Real-time PCR检测肝特异性基因均有不同程度的表达上调，以白蛋白（ALB）及转铁蛋白（TF）最显著，分别为118%及250%，差异有统计学意义（t＝7.418、5.131，P＝0.002、0.001）；吲哚菁绿摄取实验结果显示约35%呈阳性细胞；70%细胞呈糖原PAS染色阳性；尿素合成动态检测可见诱导细胞尿素分泌量呈逐增多趋势，到达肝样细胞（iHEPs）期（第20天），每106个细胞合成的尿素氮量达到了等量正常肝细胞合成的55%，即iHEPs从生物学功能上均显示转分化肝样细胞肝功能部分接近正常肝细胞水平。结论 HNF4A与表观遗传修饰（HDACi＋DNMTi）可以联合诱导人成纤维细胞转分化为肝细胞样细胞，肝样细胞在基因表达及生物学功能部分接近正常肝脏细胞。Objective To induce the hepatocellular transdifferentiation by transfecting human hepatocytes with nuclear factor 4α （HNF4A） and performing epigenetic modification [histone deacetylase inhibitor （HDACi） and DNA methyltransferase inhibitor （DNMTi）], aimed at establishing a brand new and more efficient liver cell direct conversion method. Methods The human foreskin fibroblasts （hFFs） of 8th generation were induced reprogramming by culturing in the presence of 5-azacytidine （5-AzaC） and valproic acid （VPA）. Three days later, the cells were infected with HNF4A lentivirus and then continuously cultured in the hepatocyte culture medium for 20 days. Finally, The dynamic process of morphological changes was observed by inverted phase contrast microscope. The specific hepatocellular genes were detected by real-time quantitative polymerase chain reaction （Real-time PCR）, and the hepatocellular biological function was proven by performing indocyanine green （ICG） uptake test, periodic acid-schiff （PAS） staining and assay of urea synthesis.Results During the induction, the cells gradually changed from fusiform to epithelioid in morphology, and gradually began to overexpress liver specific gene and perform biological function. Twenty days after transdifferentiation, the hepatocyte-like cells exhibited significant hepatocellular epithelial cells change in morphology with abundant cytoplasm, large circular stained nucleus and prominent nucleoli. Real-time PCR detection indicated that most liver-specific genes were upregulated, especially albumin （ALB） and transferrin （TF）, with the difference being statistically significant （t=7.418, 5.131, P=0.002, 0.001）. In ICG uptake test, PAS staining and assay of urea synthesis, the hepatocyte-like cells revealed more hepatocellular biological functional maturation than normal fibroblasts, and a lot of them was close to the levels of normal hepatocytes in biological function with the difference being statistically significant （P=0.031, 0.0

关 键 词：肝细胞 转分化 人肝细胞核因子4α 组蛋白去乙酰化 DNA甲基化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]