The impacts of dexamethasone(Dex)and thyroid hormone T3 on the insulin-stimulated Srebp-1c expression were studied in primary rat hepatocytes. Primary hepatocytes from Sprague-Dawley rats were isolated, cultured and t...The impacts of dexamethasone(Dex)and thyroid hormone T3 on the insulin-stimulated Srebp-1c expression were studied in primary rat hepatocytes. Primary hepatocytes from Sprague-Dawley rats were isolated, cultured and treated with insulin in the presence or absence of the indicated reagents over time. The mRNA levels of indicated genes were determined using real-time PCR. Insulin treatment induced the Srebp-1c expression and suppressed the Pck1 expression in a time-dependent manner. Dex treatment alone reduced the Srebp-1c expression, whereas potentiated the insulin-induced its expression, which reached to a level that was higher than the insulin alone group. On the other hand, insulin treatment completely suppressed the Dex-induced Pck1 expression in the same cells. T3 treatment did not affect the expressions of Srebp-1c and Pck1 alone or in the presence of absence of insulin or Dex. Interestingly, insulin treatment induced the Rxrg m RNA expression level in the absence or presence of T0901317, a specific agonist for the liver X receptor. Dex and insulin mutually affect each other's ability to regulate the expression levels of hepatic genes involved in glucose and fatty acid metabolism. Insulin induced Rxrg expression in primary hepatocytes, which may contribute to the induction of Srebp-1c expression in the same cells.展开更多
Background:The liver is fundamental for keeping up the entire body’s homeostasis.The liver hepatocytes have been shown to undergo genomic instability with aging.The stability of the hepatocytes depends on its nuclear...Background:The liver is fundamental for keeping up the entire body’s homeostasis.The liver hepatocytes have been shown to undergo genomic instability with aging.The stability of the hepatocytes depends on its nuclear architecture.Calorie restriction has been shown to extend life-span favorably and this may be through the reorganization of the nuclear structure.Objective:To study the effect of cyclic feeding regime on the chromatin assembly anchored to the nuclear membrane scaffold of rat models hepatocytes nuclei.Method:Rats models underwent cyclic feeding regime,after which nuclei were isolated;then,we investigated the chromatin decondensation and nuclear membrane disintegration of the hepatocytes using fluorescence imaging methods.Results:In 60 seconds,protease decondensed the chromatin and disintegrated the nuclear membrane structure of controls.After the first fasting,the time increased to 145 seconds in 3-month-old rats.The first refeeding increased the time to 156 seconds with a further rise to 340 seconds following the second fasting,then dropped to 116 seconds by the second refeeding.20 months old rats showed 186 seconds increase in the time of chromatin decondensation and nuclear membrane disintegration after the first fasting,with a decrease to 140 seconds observed after first refeeding.The second fasting increased the time to 165 seconds,which then slightly decreased to 163 seconds after the second refeeding.Conclusion:These results show that intermittent fasting may have acted on chromatin histone interactions and the structural lamin networks of the nuclear membranes in bringing about nuclear stability,which is essential for normal cellular function.展开更多
AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion...AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.展开更多
AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to in...AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.展开更多
Hepatitis C virus(HCV)infection is a global health problem,with an estimated 170 million people being chronically infected.HCV cell entry is a complex multi-step process,involving several cellular factors that trigger...Hepatitis C virus(HCV)infection is a global health problem,with an estimated 170 million people being chronically infected.HCV cell entry is a complex multi-step process,involving several cellular factors that trigger virus uptake into the hepatocytes.The high-density lipoprotein receptor scavenger receptor class B type I,tetraspanin CD81,tight junction protein claudin-1,and occludin are the main receptors that mediate the initial step of HCV infection.In addition,the virus uses cell receptor tyrosine kinases as entry regulators,such as epidermal growth factor receptor and ephrin receptor A2.This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment,internalization,and membrane fusion,and how host cell kinases regulate virus entry.The advances of the potential antiviral agents targeting this process are introduced.展开更多
AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepa...AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen(SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.RESULTS: The freshly isolated hepatocytes with high viability(85%) were successfully immortalized using retroviral gene transfer of SV40 T. SV40 T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.展开更多
Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of ...Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of MTT to formazan salt was ex-amined. Supernatant medium was collected for analy-sis of alanine aminotransferase(ALT) and ureagene-sis.Results: The two-step collagenase perfusion yielded39±12×10~6 cells/g liver tissue with a viability of88%±2%. Fine morphology and stable urea synthesisfor one week could be achieved when hepatocytes werecultured in Hepatozyme-SFM.Conclusion: High yield of hepatocytes can be isolatedwith two-step collagenese perfusion. Hepatozyme-SFM is suitable for sustained growth of hepatocytes.展开更多
Nonalcoholic fatty liver disease(NAFLD) is the most common liver disease worldwide, affecting approximately one third of the Western world. It consists of a wide spectrum of liver disorders, ranging from fatty liver t...Nonalcoholic fatty liver disease(NAFLD) is the most common liver disease worldwide, affecting approximately one third of the Western world. It consists of a wide spectrum of liver disorders, ranging from fatty liver to nonalcoholic steatohepatitis(NASH), which consists of steatosis, ballooning injury and inflammation. Despite an alarming growth in the statistics surrounding NAFLD, there are as yet no effective therapies for its treatment. Innate immune signaling has been thought to play a significant role in initiating and augmenting hepatic inflammation, contributing to the transition from nonalcoholic fatty liver to NASH. An immune response is triggered by countless signals called damage-associated molecular patterns(DAMPs) elicited by lipid-laden and damaged hepatocytes, which are recognized by pattern recognition receptors(PRRs) on hepatic immune cells to initiate inflammatory signaling. In this editorial, in addition to summarizing innate immune signaling in NAFLD and discussing potential therapies that target innate immune pathways, we have described a recent study that demonstrated that mitochondrial DNA serves as a DAMP activating a hepatic PRR, TLR9, in mice and in the plasma of NASH patients. In addition to identifying a new ligand for TLR9 during NASH progression, the study shows that blocking TLR9 reverses NASH, paving the way for the development of future NASH therapy.展开更多
AIM:To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype(SASP).METHODS:Hydrogen peroxide treatment was used to induce senescence in the human Hep G2 hepatoc...AIM:To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype(SASP).METHODS:Hydrogen peroxide treatment was used to induce senescence in the human Hep G2 hepatocyte cell line.Senescence was confirmed by cytochemical staining for a panel of markers including Ki67,p21,heterochromatin protein 1β,and senescence-associated-β-galactosidase activity.Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction(q PCR),and conditioned media was used in proteomic analyses,a human chemokine protein array,and cell migration assays to characterise the composition and function of the hepatocyte SASP.RESULTS:Senescent hepatocytes induced classical markers of senescence(p21,heterochromatin protein1β,and senescence-associated-β-galactosidase activity);and downregulated the proliferation marker,Ki67.Hepatocyte senescence induced a 4.6-fold increase in total secreted protein(P=0.06)without major alterations in the protein profile.Senescence-induced genes were identified by microarray(Benjamini Hochbergcorrected P<0.05);and,consistent with the increase in secreted protein,gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes.The hepatocyte SASP included characteristic factors such as interleukin(IL)-8 and IL-6,as well as novel components such as SAA4,IL-32and Fibrinogen,which were validated by q PCR and/or chemokine protein array.Senescent hepatocyteconditioned medium elicited migration of inflammatory(granulocyte-macrophage colony stimulating factor,GM-CSF-derived),but not non-inflammatory(CSF-1-derived)human macrophages(P=0.022),which could contribute to a pro-inflammatory microenvironment in vivo,or facilitate the clearance of senescent cells.CONCLUSION:Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.展开更多
AIM:To examine the consequences of cellular f ibronectin(cFn)accumulation during alcohol-induced injury,and inv estigate whether increased cFn could have an effect on hepatocytes(HCs)by producing factors that could co...AIM:To examine the consequences of cellular f ibronectin(cFn)accumulation during alcohol-induced injury,and inv estigate whether increased cFn could have an effect on hepatocytes(HCs)by producing factors that could cont ribute to alcohol-induced liver injury.METHODS:HCs were isolated from rats fed a control or ethanol liquid diet for four to six weeks.Exogenous c Fn(up to 7.5 μg/mL)was added to cells cultured for 20 h,and viability(lactate dehydrogenase),apoptosis(caspase activity)and se cretion of proinflammat-ory cytokines(tumor ne c rosis fac tor alpha,TNF-α and interleukin 6,IL-6),mat rix metalloproteinases(MMPs)and their inhibitors(tissue inhibitors of metall-oproteinases,TIMPs)was det ermined.Degrad ation of iodinated cFn was det ermined over a 3 h time period in the preparations.RESULTS:cFn degradation is impaired in HCs isolated from ethanol-fed animals,leading to its accumulation in the matrix.Addition of exogenous cFn did not affect viability of HCs from control or ethanolfed animals,and apoptosis was affected only at the higher concentration.Sec retion of MMPs,TIMPs,TNF-α and IL-6,however,was increased by exogenously added cFn,with HCs from ethanolfed animals showing increased susceptibility compared to the controls.CONCLUSION:These results suggest that the elevated amounts of cFn observed in alcoholic liver injury can stimulate hepatocytes to produce factors which promote further tissue damage.展开更多
AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cry...AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.展开更多
Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC t...Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.展开更多
OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were t...OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.展开更多
AIM To evaluate a culture system for bile acidformation in primary human hepatocytes incomparison with HepG2 cells.METHODS Hepatocytes were isolated fromnormal human liver tissue and were cultured inserum-free William...AIM To evaluate a culture system for bile acidformation in primary human hepatocytes incomparison with HepG2 cells.METHODS Hepatocytes were isolated fromnormal human liver tissue and were cultured inserum-free William’s E medium.The medium wascollected and renewed every 24 h.Bile acids andtheir precursors in media were finally analysed bygas chromatography-mass spectrometry.RESULTS Cholic acid(CA)andchenodeoxycholic acid(CDCA)conjugated withglycine or taurine accounted for 70% and 25% oftotal steroids.A third of CDCA was alsoconjugated with sulphuric acid.Dexamethasoneand thyroid hormone alone or in combination didnot significantly effect bile acid formation.Theaddition of cyclosporin A(10 μmol/L)inhibited thesynthesis of CA and CDCA by about 13% and30%,respectively.CONCLUSION Isolated human hepatocytes inprimary culture behave as in the intact liver byconverting cholesterol to conjugated CA andCDCA.This is in contrast to cultured HepG2 cells,which release large amounts of bile acidprecursors and unconjugated bile acids into themedium.展开更多
BACKGROUND High mobility group box-1 (HMGB1), recognized as a representative of damageassociated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting ...BACKGROUND High mobility group box-1 (HMGB1), recognized as a representative of damageassociated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting in tissue damage. Dozens of studies have shown that HMGB1 is involved in certain diseases, but the details on how injured hepatocytes release HMGB1 need to be elicited. AIM To reveal HMGB1 release mechanism in hepatocytes undergoing oxidative stress. METHODS C57BL6/J male mice were fed a high-fat diet for 12 wk plus a single binge of ethanol to induce severe steatohepatitis. Hepatocytes treated with H2O2 were used to establish an in vitro model. Serum alanine aminotransferase, liver H2O2 content and catalase activity, lactate dehydrogenase and 8-hydroxy-2- deoxyguanosine content, nicotinamide adenine dinucleotide (NAD+) levels, and Sirtuin 1 (Sirt1) activity were detected by spectrophotometry. HMGB1 release was measured by enzyme linked immunosorbent assay. HMGB1 translocation was observed by immunohistochemistry/immunofluorescence or Western blot. Relative mRNA levels were assayed by qPCR and protein expression was detected by Western blot. Acetylated HMGB1 and poly(ADP-ribose)polymerase 1 (Parp1) were analyzed by Immunoprecipitation. RESULTS When hepatocytes were damaged, HMGB1 translocated from the nucleus to the cytoplasm because of its hyperacetylation and was passively released outside both in vivo and in vitro. After treatment with Sirt1-siRNA or Sirt1 inhibitor (EX527), the hyperacetylated HMGB1 in hepatocytes increased, and Sirt1 activity inhibited by H2O2 could be reversed by Parp1 inhibitor (DIQ). Parp1 and Sirt1 are two NAD+-dependent enzymes which play major roles in the decision of a cell to live or die in the context of stress . We showed that NAD+ depletion attributed to Parp1 activation after DNA damage was caused by oxidative stress in hepatocytes and resulted in Sirt1 activity inhibition. On the contrary, Sirt1 suppressed Parp1 by negatively regulating its gene expression and deacetylation. CONCLUSION The functional inhibition between Parp1 and Sirt1 leads to HMGB1 hyperacetylation, which leads to its translocation from the nucleus to the cytoplasm and finally outside the cell.展开更多
Cellular and mitochondrial damage can be caused by labile iron pool (LIP) and mediated by reactive oxygen species (ROS). Livers of the thalassemias have highly increased levels of LIP and ROS. Green tea extract (GTE) ...Cellular and mitochondrial damage can be caused by labile iron pool (LIP) and mediated by reactive oxygen species (ROS). Livers of the thalassemias have highly increased levels of LIP and ROS. Green tea extract (GTE) and epigallocatechin 3-gallatte (EGCG) can potentially protect liver inflammation, fibrosis and cancer due to their anti-oxidative and iron-chelating activities. We studied the effects of GTE and EGCG on intracellular LIP and ROS, and mitochondrial membrane potential (ΔΨm) in mouse hepatocyte and HepG2 cell cultures using specific fluorescent techniques. Treatment with GTE (12.5 - 25 mg/dl) and EGCG (25 - 50 μM) significantly lowered levels of ΔΨm in the mouse hepatocytes;however, combined treatment of 25 μM DFP with GTE and EGCG did not enhance the decrease of hepatic ΔΨm. The results showed that GTE and EGCG effectively removed the intracellular LIP and ROS, and relieved the mitochondria membrane collapse of the liver cells, suggesting a hepatoprotective effect of green tea extract and EGCG in the hepatocytes with iron overload. Their actions might be related to iron-chelating and free radical-scavenging capacities. Whether the effects can improve iron overload and oxidative stress in thalassemia patients remains to be seen upon further examination.展开更多
Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated...Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia(21% O2) or hypoxia(2% O2).The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry(FCM).The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope.Results The cell purity of hepatocytes was over 95%.Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability.Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm.Conclusion Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics.Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.展开更多
基金the Scientific Research Project of Wuhan Municipal Health Commission for research support to Y. Zhang (WX19Y09)。
文摘The impacts of dexamethasone(Dex)and thyroid hormone T3 on the insulin-stimulated Srebp-1c expression were studied in primary rat hepatocytes. Primary hepatocytes from Sprague-Dawley rats were isolated, cultured and treated with insulin in the presence or absence of the indicated reagents over time. The mRNA levels of indicated genes were determined using real-time PCR. Insulin treatment induced the Srebp-1c expression and suppressed the Pck1 expression in a time-dependent manner. Dex treatment alone reduced the Srebp-1c expression, whereas potentiated the insulin-induced its expression, which reached to a level that was higher than the insulin alone group. On the other hand, insulin treatment completely suppressed the Dex-induced Pck1 expression in the same cells. T3 treatment did not affect the expressions of Srebp-1c and Pck1 alone or in the presence of absence of insulin or Dex. Interestingly, insulin treatment induced the Rxrg m RNA expression level in the absence or presence of T0901317, a specific agonist for the liver X receptor. Dex and insulin mutually affect each other's ability to regulate the expression levels of hepatic genes involved in glucose and fatty acid metabolism. Insulin induced Rxrg expression in primary hepatocytes, which may contribute to the induction of Srebp-1c expression in the same cells.
基金funding agency in the public,commercial,or not-for-profit sectors.
文摘Background:The liver is fundamental for keeping up the entire body’s homeostasis.The liver hepatocytes have been shown to undergo genomic instability with aging.The stability of the hepatocytes depends on its nuclear architecture.Calorie restriction has been shown to extend life-span favorably and this may be through the reorganization of the nuclear structure.Objective:To study the effect of cyclic feeding regime on the chromatin assembly anchored to the nuclear membrane scaffold of rat models hepatocytes nuclei.Method:Rats models underwent cyclic feeding regime,after which nuclei were isolated;then,we investigated the chromatin decondensation and nuclear membrane disintegration of the hepatocytes using fluorescence imaging methods.Results:In 60 seconds,protease decondensed the chromatin and disintegrated the nuclear membrane structure of controls.After the first fasting,the time increased to 145 seconds in 3-month-old rats.The first refeeding increased the time to 156 seconds with a further rise to 340 seconds following the second fasting,then dropped to 116 seconds by the second refeeding.20 months old rats showed 186 seconds increase in the time of chromatin decondensation and nuclear membrane disintegration after the first fasting,with a decrease to 140 seconds observed after first refeeding.The second fasting increased the time to 165 seconds,which then slightly decreased to 163 seconds after the second refeeding.Conclusion:These results show that intermittent fasting may have acted on chromatin histone interactions and the structural lamin networks of the nuclear membranes in bringing about nuclear stability,which is essential for normal cellular function.
基金the National Natural Science Foundation of China,No.39570212
文摘AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.
基金Supported by the National Natural Science Foundation of China,No.81160067 and No.814600124
文摘AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.
基金Supported by Research Grants from National S and T Major Project for Infectious Diseases Control,No.2012ZX10002003-004-010National Natural Science Foundation of China,No.81171564,No.81273557 and No.81302812+2 种基金Medical Youth Science Program,No.13QNP100Shanghai Municipal Natural Science Foundation,No.13ZR1449300Shanghai LAD Project,No.B901
文摘Hepatitis C virus(HCV)infection is a global health problem,with an estimated 170 million people being chronically infected.HCV cell entry is a complex multi-step process,involving several cellular factors that trigger virus uptake into the hepatocytes.The high-density lipoprotein receptor scavenger receptor class B type I,tetraspanin CD81,tight junction protein claudin-1,and occludin are the main receptors that mediate the initial step of HCV infection.In addition,the virus uses cell receptor tyrosine kinases as entry regulators,such as epidermal growth factor receptor and ephrin receptor A2.This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment,internalization,and membrane fusion,and how host cell kinases regulate virus entry.The advances of the potential antiviral agents targeting this process are introduced.
基金Supported by Major Scientific and Technological Project of Shandong Province,No.201221019Cisco Clinical Oncology Research Fund and Bayer Schering Cancer Research Fund,No.Y-B2012-011
文摘AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen(SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.RESULTS: The freshly isolated hepatocytes with high viability(85%) were successfully immortalized using retroviral gene transfer of SV40 T. SV40 T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.
文摘Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of MTT to formazan salt was ex-amined. Supernatant medium was collected for analy-sis of alanine aminotransferase(ALT) and ureagene-sis.Results: The two-step collagenase perfusion yielded39±12×10~6 cells/g liver tissue with a viability of88%±2%. Fine morphology and stable urea synthesisfor one week could be achieved when hepatocytes werecultured in Hepatozyme-SFM.Conclusion: High yield of hepatocytes can be isolatedwith two-step collagenese perfusion. Hepatozyme-SFM is suitable for sustained growth of hepatocytes.
文摘Nonalcoholic fatty liver disease(NAFLD) is the most common liver disease worldwide, affecting approximately one third of the Western world. It consists of a wide spectrum of liver disorders, ranging from fatty liver to nonalcoholic steatohepatitis(NASH), which consists of steatosis, ballooning injury and inflammation. Despite an alarming growth in the statistics surrounding NAFLD, there are as yet no effective therapies for its treatment. Innate immune signaling has been thought to play a significant role in initiating and augmenting hepatic inflammation, contributing to the transition from nonalcoholic fatty liver to NASH. An immune response is triggered by countless signals called damage-associated molecular patterns(DAMPs) elicited by lipid-laden and damaged hepatocytes, which are recognized by pattern recognition receptors(PRRs) on hepatic immune cells to initiate inflammatory signaling. In this editorial, in addition to summarizing innate immune signaling in NAFLD and discussing potential therapies that target innate immune pathways, we have described a recent study that demonstrated that mitochondrial DNA serves as a DAMP activating a hepatic PRR, TLR9, in mice and in the plasma of NASH patients. In addition to identifying a new ligand for TLR9 during NASH progression, the study shows that blocking TLR9 reverses NASH, paving the way for the development of future NASH therapy.
基金Supported by the National Health and Medical Research Council of Australia(NHMRC),APP1044650 and APP1003108the Queensland Government’s Smart State Health and Medical Research Fund+5 种基金the Princess Alexandra Hospital Research and Development Foundation and The Australian Liver FoundationIrvine KM is the recipient of the Australian Liver Foundation Pauline Hall FellowshipPowell EE is the recipient of an NHMRC Practitioner Fellowship,APP1004242Sweet MJ is the recipient of an Australian Research Council(ARC)Future Fellowship,FT100100657an honorary NHMRC Senior Research Fellowship,APP1003470Hill MM is the recipient of an ARC Future Fellowship,FT120100251
文摘AIM:To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype(SASP).METHODS:Hydrogen peroxide treatment was used to induce senescence in the human Hep G2 hepatocyte cell line.Senescence was confirmed by cytochemical staining for a panel of markers including Ki67,p21,heterochromatin protein 1β,and senescence-associated-β-galactosidase activity.Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction(q PCR),and conditioned media was used in proteomic analyses,a human chemokine protein array,and cell migration assays to characterise the composition and function of the hepatocyte SASP.RESULTS:Senescent hepatocytes induced classical markers of senescence(p21,heterochromatin protein1β,and senescence-associated-β-galactosidase activity);and downregulated the proliferation marker,Ki67.Hepatocyte senescence induced a 4.6-fold increase in total secreted protein(P=0.06)without major alterations in the protein profile.Senescence-induced genes were identified by microarray(Benjamini Hochbergcorrected P<0.05);and,consistent with the increase in secreted protein,gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes.The hepatocyte SASP included characteristic factors such as interleukin(IL)-8 and IL-6,as well as novel components such as SAA4,IL-32and Fibrinogen,which were validated by q PCR and/or chemokine protein array.Senescent hepatocyteconditioned medium elicited migration of inflammatory(granulocyte-macrophage colony stimulating factor,GM-CSF-derived),but not non-inflammatory(CSF-1-derived)human macrophages(P=0.022),which could contribute to a pro-inflammatory microenvironment in vivo,or facilitate the clearance of senescent cells.CONCLUSION:Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.
基金Supported by the National Institute on Alcohol Abuse and Alcoholism and the US Department of Veterans Affairs
文摘AIM:To examine the consequences of cellular f ibronectin(cFn)accumulation during alcohol-induced injury,and inv estigate whether increased cFn could have an effect on hepatocytes(HCs)by producing factors that could cont ribute to alcohol-induced liver injury.METHODS:HCs were isolated from rats fed a control or ethanol liquid diet for four to six weeks.Exogenous c Fn(up to 7.5 μg/mL)was added to cells cultured for 20 h,and viability(lactate dehydrogenase),apoptosis(caspase activity)and se cretion of proinflammat-ory cytokines(tumor ne c rosis fac tor alpha,TNF-α and interleukin 6,IL-6),mat rix metalloproteinases(MMPs)and their inhibitors(tissue inhibitors of metall-oproteinases,TIMPs)was det ermined.Degrad ation of iodinated cFn was det ermined over a 3 h time period in the preparations.RESULTS:cFn degradation is impaired in HCs isolated from ethanol-fed animals,leading to its accumulation in the matrix.Addition of exogenous cFn did not affect viability of HCs from control or ethanolfed animals,and apoptosis was affected only at the higher concentration.Sec retion of MMPs,TIMPs,TNF-α and IL-6,however,was increased by exogenously added cFn,with HCs from ethanolfed animals showing increased susceptibility compared to the controls.CONCLUSION:These results suggest that the elevated amounts of cFn observed in alcoholic liver injury can stimulate hepatocytes to produce factors which promote further tissue damage.
基金Supported by Grants from VINNMER,Lundin foundation, R&D Funds from Stockholm County and Karolinska Institutet (ALF),and the Swedish Research Council
文摘AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.
基金Supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science,No.17K08966(to Ohkoshi S)
文摘Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.
基金supported by National Natural Science Foundation of China(81700523)
文摘OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.
基金the Swedish Medical Research Council(03X-4793 and 03X-7890)
文摘AIM To evaluate a culture system for bile acidformation in primary human hepatocytes incomparison with HepG2 cells.METHODS Hepatocytes were isolated fromnormal human liver tissue and were cultured inserum-free William’s E medium.The medium wascollected and renewed every 24 h.Bile acids andtheir precursors in media were finally analysed bygas chromatography-mass spectrometry.RESULTS Cholic acid(CA)andchenodeoxycholic acid(CDCA)conjugated withglycine or taurine accounted for 70% and 25% oftotal steroids.A third of CDCA was alsoconjugated with sulphuric acid.Dexamethasoneand thyroid hormone alone or in combination didnot significantly effect bile acid formation.Theaddition of cyclosporin A(10 μmol/L)inhibited thesynthesis of CA and CDCA by about 13% and30%,respectively.CONCLUSION Isolated human hepatocytes inprimary culture behave as in the intact liver byconverting cholesterol to conjugated CA andCDCA.This is in contrast to cultured HepG2 cells,which release large amounts of bile acidprecursors and unconjugated bile acids into themedium.
基金Supported by the National Natural Science Foundation of China,No.81503367 and No.81703832
文摘BACKGROUND High mobility group box-1 (HMGB1), recognized as a representative of damageassociated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting in tissue damage. Dozens of studies have shown that HMGB1 is involved in certain diseases, but the details on how injured hepatocytes release HMGB1 need to be elicited. AIM To reveal HMGB1 release mechanism in hepatocytes undergoing oxidative stress. METHODS C57BL6/J male mice were fed a high-fat diet for 12 wk plus a single binge of ethanol to induce severe steatohepatitis. Hepatocytes treated with H2O2 were used to establish an in vitro model. Serum alanine aminotransferase, liver H2O2 content and catalase activity, lactate dehydrogenase and 8-hydroxy-2- deoxyguanosine content, nicotinamide adenine dinucleotide (NAD+) levels, and Sirtuin 1 (Sirt1) activity were detected by spectrophotometry. HMGB1 release was measured by enzyme linked immunosorbent assay. HMGB1 translocation was observed by immunohistochemistry/immunofluorescence or Western blot. Relative mRNA levels were assayed by qPCR and protein expression was detected by Western blot. Acetylated HMGB1 and poly(ADP-ribose)polymerase 1 (Parp1) were analyzed by Immunoprecipitation. RESULTS When hepatocytes were damaged, HMGB1 translocated from the nucleus to the cytoplasm because of its hyperacetylation and was passively released outside both in vivo and in vitro. After treatment with Sirt1-siRNA or Sirt1 inhibitor (EX527), the hyperacetylated HMGB1 in hepatocytes increased, and Sirt1 activity inhibited by H2O2 could be reversed by Parp1 inhibitor (DIQ). Parp1 and Sirt1 are two NAD+-dependent enzymes which play major roles in the decision of a cell to live or die in the context of stress . We showed that NAD+ depletion attributed to Parp1 activation after DNA damage was caused by oxidative stress in hepatocytes and resulted in Sirt1 activity inhibition. On the contrary, Sirt1 suppressed Parp1 by negatively regulating its gene expression and deacetylation. CONCLUSION The functional inhibition between Parp1 and Sirt1 leads to HMGB1 hyperacetylation, which leads to its translocation from the nucleus to the cytoplasm and finally outside the cell.
文摘Cellular and mitochondrial damage can be caused by labile iron pool (LIP) and mediated by reactive oxygen species (ROS). Livers of the thalassemias have highly increased levels of LIP and ROS. Green tea extract (GTE) and epigallocatechin 3-gallatte (EGCG) can potentially protect liver inflammation, fibrosis and cancer due to their anti-oxidative and iron-chelating activities. We studied the effects of GTE and EGCG on intracellular LIP and ROS, and mitochondrial membrane potential (ΔΨm) in mouse hepatocyte and HepG2 cell cultures using specific fluorescent techniques. Treatment with GTE (12.5 - 25 mg/dl) and EGCG (25 - 50 μM) significantly lowered levels of ΔΨm in the mouse hepatocytes;however, combined treatment of 25 μM DFP with GTE and EGCG did not enhance the decrease of hepatic ΔΨm. The results showed that GTE and EGCG effectively removed the intracellular LIP and ROS, and relieved the mitochondria membrane collapse of the liver cells, suggesting a hepatoprotective effect of green tea extract and EGCG in the hepatocytes with iron overload. Their actions might be related to iron-chelating and free radical-scavenging capacities. Whether the effects can improve iron overload and oxidative stress in thalassemia patients remains to be seen upon further examination.
基金funded by National Natural Science Foundation [81370719 and 81671535]Jiangsu Science Foundation [BE2015642]+3 种基金Jiangsu Key Discipline of Human Assisted Reproduction Medicine Foundation [FXK201749]Jiangsu Provincial Medical Talent of the Project of Invigorating Healthcare through Science,Technology and Education [ZDRCA2016044]and Chinese Medical Association Clinical Medicine Research Reproductive Medicine [17020270696]The Priority Academic Program Development of the Jiangsu Higher Education Institutes(PAPD)
文摘Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia(21% O2) or hypoxia(2% O2).The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry(FCM).The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope.Results The cell purity of hepatocytes was over 95%.Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability.Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm.Conclusion Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics.Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.
