小鼠第13.5天胚肝细胞诱导HepG2细胞分化的作用机制  

The effect and mechanism of differentiation of HepG2 cells induced by mouse embryonic hepatocytes at day 13.5 of gestation

作　　者：肖志刚 郑丽 王梓瀛[1] 刘昆 王瑜 齐锦生 栗彦宁 XIAO Zhigang;ZHENG Li;WANG Ziying;LIU Kun;WANG Yu;QI Jinsheng;LI Yanning(College of Integrated Chinese and Western Medicine of Hebei Medical University,Shijiazhuang 050017,China;Hebei Key Lab of Laboratory Animal Science of Hebei Medical University,Shijiazhuang 050017;Xingtai Medical College,Xingtai 054000)

机构地区：[1]河北医科大学中西医结合学院,石家庄050017 [2]河北医科大学实验动物学部,石家庄050017 [3]邢台医学高等专科学校,河北邢台054000

出　　处：《中国比较医学杂志》2022年第4期47-53,共7页Chinese Journal of Comparative Medicine

基　　金：河北省自然科学基金(H2020206328,H2020206386);邢台市科技局自筹项目(2018ZC133)。

摘　　要：目的探讨小鼠第13.5天胚肝细胞是否通过抑制β-Catenin诱导人肝细胞肝癌细胞株HepG2细胞分化及其作用机制。方法用免疫荧光法检测肝细胞标记分子-白蛋白(ALB)的分布,以鉴定小鼠第13.5天胚肝细胞。小鼠第13.5天胚肝细胞与HepG2细胞株共培养24 h、48 h后,采用qRT-PCR方法检测HepG2细胞株中肝癌标记分子-甲胎蛋白(AFP)、肝细胞分化调控因子-肝细胞核因子4α(HNF-4α)和成熟肝细胞标记分子-细胞色素P450家族成员1B1(CYP1B1)和乙醇脱氢酶1C(ADH1C)的mRNA表达。共培养48 h后,观察HepG2细胞株形态变化,采用Western blot法检测AFP、HNF-4α和β-Catenin的蛋白含量,免疫荧光法检测β-Catenin蛋白在细胞的分布。应用β-Catenin抑制剂处理后,观察HepG2细胞株形态变化并且检测AFP蛋白表达。结果原代培养细胞ALB荧光显示阳性。与对照组相比,共培养后HepG2细胞株中,AFP相对mRNA表达量在24 h和48 h均显著降低,HNF-4α、CYP1B1和ADH1C的相对mRNA表达量在24 h和48 h均显著升高。与对照组相比,共培养48 h后,HepG2细胞株生长明显抑制,细胞形态趋于正常肝细胞的六边形,AFP和HNF-4α蛋白表达明显升高。与对照组相比,共培养48 h后,β-Catenin的蛋白含量明显降低,同时HepG2细胞株中β-Catenin的荧光明显减弱。与对照组相比,β-Catenin抑制剂处理可明显改变HepG2细胞株形态,引起共培养组HepG2细胞株形态更剧烈的变化,显著抑制AFP的蛋白表达。结论小鼠第13.5天胚肝细胞可能主要通过抑制β-Catenin,诱导HepG2细胞株分化。Objective To explore the differentiation of HepG2 cells induced by mouse embryonic hepatocytes at day 13.5 of gestation through the inhibition ofβ-Catenin.Methods The distribution of hepatocyte marker moleculealbumin(ALB)was detected by immunofluorescence to identify the hepatocytes of mouse embryo on day 13.5.After HepG2 cells were co-cultured with embryonic hepatocytes at day 13.5 of gestation for 24 h and 48 h,the mRNA expressions of AFP(biomarker of hepatocellular carcinoma),HNF-4α(controller of hepatocyte differentiation),CYP1B1(biomarker of mature hepatocytes)and ADH1C(another biomarker of mature hepatocytes)were measured by reverse transcriptional quantitative polymerase chain reaction(RT-qPCR).After HepG2 cells were co-cultured with day 13.5 of gestation embryonic hepatocytes for 48 h,the morphology was observed;AFP,HNF-4αandβ-catenin expressions were measured by Western blot andβ-Catenin distribution was determined by immunofluorescence.Following treatment with theβ-catenin inhibitor,morphology was observed and AFP content was measured.Results Most cells were ALB-positive.The relative mRNA expression of AFP decreased significantly after 24 h and 48 h co-culture,and HNF-4α,CYP1B1 and ADH1C increased significantly at 24 h and 48 h co-culture compared with the levels in the controls.The proliferation of HepG2 cells was suppressed and cell morphology tended to be hexagonal like normal hepatocytes;AFP protein content decreased and HNF-4αprotein content increased after co-culture with embryonic hepatocytes at day 13.5 of gestation for 48 h.Compared with the levels in the control,β-Catenin protein content decreased in the co-culture group andβ-catenin was markedly attenuated in the co-cultured HepG2 cells in immunofluorescence result.Compared with the effects in the control group,β-catenin inhibitor treatment caused notable morphological changes of HepG2 cells and induced more dramatic morphological changes of HepG2 cells;furthermore,it reduced AFP protein content.Conclusions HepG2 cells may be induc

关 键 词：HEPG2 细胞分化 胚肝细胞 Β-CATENIN HNF-4Α 

分 类 号：R-33[医药卫生]