缺氧诱导因子-1α通过调控蛋白激酶B/核因子-κB信号通路促进肝癌细胞干细胞标志物CD133的表达  

Hypoxia inducible factor-1αpromotes the expression of stem cell marker CD133 in hepatoma cells by regulating protein kinase B/nuclear factor-κB signaling pathway

作　　者：赵金金 崔非非 莫清江[1] 汪磊[1] 林志强[1] 张海光 焦路阳[1] ZHAO Jinjin;CUI Feifei;MO Qingjiang;WANG Lei;LIN Zhiqiang;ZHANG Haiguang;JIAO Luyang(Department of Clinical Laboratory,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;Department of Obsterics and Gynecology,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)

机构地区：[1]新乡医学院第一附属医院检验科,河南卫辉453100 [2]新乡医学院第一附属医院妇产科,河南卫辉453100

出　　处：《新乡医学院学报》2022年第4期301-307,共7页Journal of Xinxiang Medical University

基　　金：新乡医学院第一附属医院博士科研项目启动基金(编号:10243);河南省医学科技攻关(联合共建)项目(编号:LHGJ20190453);新乡医学院第一附属医院青年培育基金项目(编号:QN-2020-B01)。

摘　　要：目的探讨缺氧诱导因子-1α(HIF-1α)诱导肝癌细胞中干细胞标志物CD133表达的分子机制及意义。方法将HepG2细胞随机分为空白对照组、对照组和实验组,空白对照组HepG2细胞不做转染,对照组和实验组HepG2细胞分别转染空载体慢病毒pHBLV-ZsGreen-Puro和HIF-1α高表达慢病毒pHBLV-HIF1A-3flag-ZsGreen-Puro。使用荧光显微镜观察对照组和实验组细胞中绿色荧光蛋白(GFP)的表达,并计算转染效率;采用Western blot法检测3组细胞中HIF-1α蛋白及蛋白激酶B(Akt)/核因子-κB(NF-κB)信号通路蛋白磷酸化蛋白激酶B(p-Akt)、Akt、p-p65、p65蛋白的相对表达量,流式细胞术检测对照组和实验组CD133阳性细胞率。另取实验组细胞随机分为HepG2-HIF-1α组、Akt激酶抑制剂组和NF-κB信号通路抑制剂组,Akt激酶抑制剂组和NF-κB信号通路抑制剂组细胞分别给予终浓度为0.4、0.8、1.6、3.2、6.3、12.5、25.0μmol·L^(-1) Akt激酶抑制剂及终浓度为3.0、6.0、12.5、25.0、50.0、100.0、200.0、400.0μmol·L^(-1) NF-κB信号通路抑制剂干预;采用四甲基偶氮唑盐法检测Akt激酶抑制剂组和NF-κB信号通路抑制剂组细胞活力,流式细胞术检测HepG2-HIF-1α组、Akt激酶抑制剂组和NF-κB信号通路抑制剂组CD133阳性细胞率的变化。结果对照组和实验组GFP阳性细胞比例显著高于空白对照组(P<0.05);对照组与实验组GFP阳性细胞比例比较差异无统计学意义(P>0.05)。实验组细胞中HIF-1α相对表达量显著高于空白对照组及对照组(P<0.05);空白对照组与对照组细胞中HIF-1α相对表达量比较差异无统计学意义(P>0.05)。对照组和实验组CD133阳性细胞率分别为(4.42±0.29)%、(15.43±0.41)%,实验组CD133阳性细胞率显著高于对照组(t=22.160,P<0.05)。实验组细胞中p-Akt/Akt、p-p65/p65显著高于对照组(P<0.05)。0.4、0.8、1.6、3.2、6.3、12.5、25.0μmol·L^(-1) Akt激酶抑制剂组细胞活力比较差异无统计学意义(F=1.30Objective To investigate the mechanism and significance of the expression of stem cell marker CD133 in hepatoma cells induced by hypoxia inducible factor-1α(HIF-1α).Methods HepG2 cells were randomly divided into blank control group,control group and experimental group;HepG2 cells in the blank control group were not transfected,the cells in the control group and experimental group were transfected with empty vector lentivirus pHBLV-ZsGreen-Puro or HIF-1a high expression lentivirus pHBLV-HIF1A-3flag-ZsGreen-Puro,respectively.The transfection efficiency of cells in the control group and experimental group was confirmed by the expression of green fluorescent protein(GFP)through fluorescence microscope;the relative expressions of HIF-1a and phosphorylated protein kinase B(p-Akt),protein kinase B(Akt),p-p65 and p65 protein in Akt/nuclear factor-κB(NF-κB)pathway in the three groups were detected by Western blot;the rarte of CD133 positive cells in the control group and experimental group were detected by flow cytometry.In addition,the cells in the experimental group were randomly divided into HepG2-HIF-1αgroup,Akt kinase inhibitor group and NF-κB signal pathway inhibitor group;the cells in the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group were treated with the final concentration of 0.4,0.8,1.6,3.2,6.3,12.5,25.0μmol·L^(-1) Akt kinase inhibitor or 3.0,6.0,12.5,25.0,50.0,100.0,200.0,400.0μmol·L^(-1) NF-κB signal pathway inhibitor;the cell viability in the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group was detected by tetramethylazozole salt assay.The rate of CD133 positive cells in the three groups was detected by flow cytometry.Results The proportion of GFP positive cells in the control group and experiment group was significantly higher than that in the blank control group(P<0.05);there was no significant difference in the proportion of GFP positive cells between the control group and the experimental group(P>0.05).The relative expression of HIF-1a in the experimen

关 键 词：肝癌 缺氧诱导因子-1Α 蛋白激酶B 核因子-ΚB 干细胞标志物 CD133 

分 类 号：R735.7[医药卫生—肿瘤]