BACKGROUND Senescence is characterized by a decline in hepatocyte function,with impairment of metabolism and regenerative capacity.Several models that duplicate liver functions in vitro are essential tools for studyin...BACKGROUND Senescence is characterized by a decline in hepatocyte function,with impairment of metabolism and regenerative capacity.Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism,liver diseases,and organ regeneration.The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors.However,the impact of senescence on HepaRG cells is not yet known.AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.METHODS We compared the transdifferentiation capacity of cells over 10(passage 10[P10])vs P20.Aging was evaluated by senescence-associated(SA)beta-galactosidase activity and the comet assay.HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry(expression of cluster of differentiation 49a[CD49a],CD49f,CD184,epithelial cell adhesion molecule[EpCAM],and cytokeratin 19[CK19]),quantitative PCR analysis(expression of albumin,cytochrome P4503A4[CYP3A4],γ-glutamyl transpeptidase[γ-GT],and carcinoembryonic antigen[CEA]),and functional analyses(albumin secretion,CYP3A4,andγ-GT).Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide(NAD^(+))/NAD with hydrogen(NADH)content were also measured.RESULTS SAβ-galactosidase staining was higher in P20 than P10 HepaRG cells;in parallel,the comet assay showed consistent DNA damage in P20 HepaRG cells.With respect to P10,P20 HepaRG cells exhibited a reduction of CD49a,CD49f,CD184,EpCAM,and CK19 after the induction of transdifferentiation.Furthermore,lower gene expression of albumin,CYP3A4,andγ-GT,as well as reduced albumin secretion capacity,CYP3A4,andγ-GT activity were reported in transdifferentiated P20 compared to P10 cells.By contrast,the gene expression level of CEA was not reduced by transdifferentiation in P20 cells.Of note,both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells.Finally,both ATP and NAD^(+)/NADH were depleted in P20 cells with respect to P10 cells.CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells,with consequent impairment of metabolic and regenerative properties,which may alter applications in basic studies.展开更多
Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds.All members of this family can cause acute and chronic hepatic infec-tio...Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds.All members of this family can cause acute and chronic hepatic infec-tion,which in the case of human hepatitis B virus(HBV) constitutes a major global health problem.Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades,the mechanisms of attachment and productive entrance into the differentiated host hepa-tocytes are still enigmatic.The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems.Thus,for more than twenty years,differentiated primary hepato-cytes from the respective species were the only in vitro models for both orthohepadnaviruses(e.g.HBV) and avihepadnaviruses(e.g.duck hepatitis B virus [DHBV]).Two important discoveries have been made recently re-garding HBV:(1) primary hepatocytes from tree-shrews;i.e.,Tupaia belangeri,can be substituted for primary hu-man hepatocytes,and(2) a human hepatoma cell line(HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro.A number of potential HBV receptor candidates have been described in the past,but none of them have been confirmed to function as a receptor.For DHBV and prob-ably all other avian hepadnaviruses,carboxypeptidase D(CPD) has been shown to be indispensable for infection,although the exact role of this molecule is still under debate.While still restricted to the use of primary duck hepatocytes(PDH),investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection.However,with emerging dataobtained from the new HBV infection systems,the hope that DHBV utilizes the same mechanism as HBV only partially held true.Nevertheless,both HBV and DHBV in vitro infection systems will help to:(1) functionally dis-sect the hepadnaviral entry pathways,(2) perform re-verse genetics(e.g.test the fitness of escape mutants),(3) titrate and map neutralizing antibodies,(4) improve current vaccines to combat acute and chronic infections of hepatitis B,and(5) develop entry inhibitors for future clinical applications.展开更多
消癌平注射液(XAP-Ⅰ)提取自通关藤,在我国临床上广泛用于治疗各种癌症,而从其复杂组分中阐释生物活性成分一直是学界研究的热点内容。本文结合三重四级杆质谱仪中的全扫描(full scan,FS)、母离子扫描(precursor ion scan,PCIS)、子离...消癌平注射液(XAP-Ⅰ)提取自通关藤,在我国临床上广泛用于治疗各种癌症,而从其复杂组分中阐释生物活性成分一直是学界研究的热点内容。本文结合三重四级杆质谱仪中的全扫描(full scan,FS)、母离子扫描(precursor ion scan,PCIS)、子离子扫描(product ion,PDIS)及多反应监测(multiple reaction monitoring,MRM)模式鉴定了XAP-Ⅰ中的主要孕甾烷糖苷及酚酸类成分,可利用丰富的加合离子信息确定组分结构特性并实现高灵敏度分析。此外,本文应用定向细胞结合植物化学组分筛选策略以发现XAP-Ⅰ中的生物活性成分,该策略可实现对XAP-Ⅰ的高通量定性、定量分析,并显著提高XAP-Ⅰ潜在生物活性成分筛选的灵敏度及选择性。展开更多
文摘BACKGROUND Senescence is characterized by a decline in hepatocyte function,with impairment of metabolism and regenerative capacity.Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism,liver diseases,and organ regeneration.The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors.However,the impact of senescence on HepaRG cells is not yet known.AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.METHODS We compared the transdifferentiation capacity of cells over 10(passage 10[P10])vs P20.Aging was evaluated by senescence-associated(SA)beta-galactosidase activity and the comet assay.HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry(expression of cluster of differentiation 49a[CD49a],CD49f,CD184,epithelial cell adhesion molecule[EpCAM],and cytokeratin 19[CK19]),quantitative PCR analysis(expression of albumin,cytochrome P4503A4[CYP3A4],γ-glutamyl transpeptidase[γ-GT],and carcinoembryonic antigen[CEA]),and functional analyses(albumin secretion,CYP3A4,andγ-GT).Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide(NAD^(+))/NAD with hydrogen(NADH)content were also measured.RESULTS SAβ-galactosidase staining was higher in P20 than P10 HepaRG cells;in parallel,the comet assay showed consistent DNA damage in P20 HepaRG cells.With respect to P10,P20 HepaRG cells exhibited a reduction of CD49a,CD49f,CD184,EpCAM,and CK19 after the induction of transdifferentiation.Furthermore,lower gene expression of albumin,CYP3A4,andγ-GT,as well as reduced albumin secretion capacity,CYP3A4,andγ-GT activity were reported in transdifferentiated P20 compared to P10 cells.By contrast,the gene expression level of CEA was not reduced by transdifferentiation in P20 cells.Of note,both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells.Finally,both ATP and NAD^(+)/NADH were depleted in P20 cells with respect to P10 cells.CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells,with consequent impairment of metabolic and regenerative properties,which may alter applications in basic studies.
基金Supported by grant SFB535/A2 from DFG, EU QLK 2000- 01476 and DFG UR72/1-3, UR72/1-4
文摘Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds.All members of this family can cause acute and chronic hepatic infec-tion,which in the case of human hepatitis B virus(HBV) constitutes a major global health problem.Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades,the mechanisms of attachment and productive entrance into the differentiated host hepa-tocytes are still enigmatic.The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems.Thus,for more than twenty years,differentiated primary hepato-cytes from the respective species were the only in vitro models for both orthohepadnaviruses(e.g.HBV) and avihepadnaviruses(e.g.duck hepatitis B virus [DHBV]).Two important discoveries have been made recently re-garding HBV:(1) primary hepatocytes from tree-shrews;i.e.,Tupaia belangeri,can be substituted for primary hu-man hepatocytes,and(2) a human hepatoma cell line(HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro.A number of potential HBV receptor candidates have been described in the past,but none of them have been confirmed to function as a receptor.For DHBV and prob-ably all other avian hepadnaviruses,carboxypeptidase D(CPD) has been shown to be indispensable for infection,although the exact role of this molecule is still under debate.While still restricted to the use of primary duck hepatocytes(PDH),investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection.However,with emerging dataobtained from the new HBV infection systems,the hope that DHBV utilizes the same mechanism as HBV only partially held true.Nevertheless,both HBV and DHBV in vitro infection systems will help to:(1) functionally dis-sect the hepadnaviral entry pathways,(2) perform re-verse genetics(e.g.test the fitness of escape mutants),(3) titrate and map neutralizing antibodies,(4) improve current vaccines to combat acute and chronic infections of hepatitis B,and(5) develop entry inhibitors for future clinical applications.
基金The Natural Science Foundation of Gansu Province (Grant No. 21JR7RA353)the First Hospital of Lanzhou University Science Foundations (Grant No. ldyyyn2018-08)the Research Foundation of Education Bureau of Gansu Province,China (Grant No. 2020B-001)。
文摘消癌平注射液(XAP-Ⅰ)提取自通关藤,在我国临床上广泛用于治疗各种癌症,而从其复杂组分中阐释生物活性成分一直是学界研究的热点内容。本文结合三重四级杆质谱仪中的全扫描(full scan,FS)、母离子扫描(precursor ion scan,PCIS)、子离子扫描(product ion,PDIS)及多反应监测(multiple reaction monitoring,MRM)模式鉴定了XAP-Ⅰ中的主要孕甾烷糖苷及酚酸类成分,可利用丰富的加合离子信息确定组分结构特性并实现高灵敏度分析。此外,本文应用定向细胞结合植物化学组分筛选策略以发现XAP-Ⅰ中的生物活性成分,该策略可实现对XAP-Ⅰ的高通量定性、定量分析,并显著提高XAP-Ⅰ潜在生物活性成分筛选的灵敏度及选择性。
