Research progress on the development of hepatocyte growth factor/c-Met signaling pathway in gastric cancer:A review

Hepatocyte growth factor(HGF)and its receptor,c-Met,play important roles in the occurrence,development,and treatment of gastric cancer(GC).This review explored the function of the HGF/c-Met signaling pathway in GC and its potential targeted therapeutic mechanisms.As one of the most common malignant tumors worldwide,GC has a complex pathogenesis and limited therapeutic options.Therefore,a thorough understanding of the molecular mechanism of GC is very important for the development of new therapeutic methods.The HGF/c-Met signaling pathway plays an important role in the proliferation,migration,and invasion of GC cells and has become a new therapeutic target.This review summarizes the current research progress on the role of HGF/c-Met in GC and discusses targeted therapeutic strategies targeting this signaling pathway,providing new ideas and directions for the treatment of GC.

Hepatocyte growth factor promotes retinal pigment epithelium cell activity through MET/AKT signaling pathway

AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepithelial transition factor(MET)inhibitor SU11274 in vitro.Cell viability was detected by a Cell Counting Kit-8 assay.Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay,respectively.The expression levels of MET,phosphorylated MET,protein kinase B(AKT),and phosphorylated AKT proteins were determined by Western blot assay.The MET and phosphorylated MET proteins were also determined by immunofluorescence assay.RESULTS:HGF increased ARPE-19 cells’viability,proliferation and migration,and induced an increase of phosphorylated MET and phosphorylated AKT proteins.SU11274 significantly reduced cell viability,proliferation,and migration and decreased the expression of MET and AKT proteins.SU11274 suppressed HGF-induced increase of viability,proliferation,and migration in ARPE-19 cells.Additionally,SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins.CONCLUSION:HGF enhances cellular viability,proliferation,and migration in RPE cells through the MET/AKT signaling pathway,whereas this enhancement is suppressed by the MET inhibitor SU11274.HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Effect of hepatocyte growth factor on inflammatory factors associated with CCL_(4)-induced hepatocyte injury

Objective:The aim of this study is to investigate the effects of Hepatocyte Growth Factor(HGF)on the expression levels of IL-8,TNF-α,IL-4,and IL-21 in mice with liver injury induced by CCL_(4).Methods:An acute liver injury mouse model was established using CCL_(4),and hepatocytes and white blood cells were separated by gradient density centrifugation.Different concentrations of HGF were added in vitro,and the expression levels of cytokines were detected using ELISA.Results:In the in vivo injury model,the hepatocyte experiment results showed that the expression level of IL-8 was reduced in the 10 ng/mL HGF group compared to the injured hepatocyte group(P<0.05),and increased in the 50 ng/mL HGF group compared to the 10 ng/mL HGF group(P<0.05).For IL-4,the expression levels were reduced in both the 25 ng/mL HGF group(P<0.05)and the 50 ng/mL HGF group(P<0.05)compared to the injured hepatocyte group.The white blood cell experiment results showed that the expression levels of TNF-αwere reduced in both the 10ng/ml HGF group(P<0.05)and the 25 ng/mL HGF group(P<0.05)compared to the injured white blood cell group.In the in vitro injury model,hepatocyte experiment results showed that the expression levels of TNF-αwere reduced in both the 25 ng/mL HGF group(P<0.05)and the 50 ng/mL HGF group(P<0.05)compared to the normal control group.For IL-4,the expression level was reduced in the 25 ng/mL HGF group compared to the normal control group(P<0.05).The white blood cell experiment results showed that the expression level of TNF-αwas increased in the 50 ng/mL HGF group compared to the 10 ng/mL HGF group(P<0.001);for IL-21,the expression levels were reduced in the CCL_(4) model group(P<0.05),10 ng/mL HGF group(P<0.05),25 ng/mL HGF group(P<0.05),and 50 ng/mL HGF group(P<0.05)compared to the normal control group.Conclusion:when the liver of mice is acutely damaged by CCL_(4),HGF can reduce the expression levels of inflammatory cytokines IL-8,TNF-α,IL-4 in hepatocytes,and TNF-αin liver white blood cells.

Dexamethasone potentiates the insulin-induced Srebp-1c expression in primary rat hepatocytes

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.

Cyclic feeding regime may delay aging in animals by enhancing the hepatocytes nuclei structure

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.

Purification and Characterization of Hepatocyte Regeneration Stimulatory Factor from Shark Liver

Aim To purify hepatocyte regeneration stimulatory factor from shark liver and research its molecular feature and activity. Methods and Results Hepatocyte regeneration stimulatory factor (sHRSF) was isolated from healthy shark livers and separated by homogenization, freezing melting, heat treating, centrifugation, and ultrafiltration. HRSF activity was found mainly in the subfraction of molecular weight less than 30 000 daltons. This crude ultrafiltrate was further purified successively by DEAE Sepharose fast flow chromatography, FPLC Resource 30Q, Resource Q and Mono Q chromatography. A single band was displayed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to molecular weight of 14 600 daltons. The characteristic absorption was obtained at the wavelength 276 nm. The isoelectric point was about 5 1. It contained 18 amino acids and the 15 N terminal amino acid residues were LVGPIGAVGPAGKDG. It had a significant activity in stimulating liver to regenerate. Conclusion We obtained an unknown new active protein, that is hepatocyte regeneration stimulatory factor from shark liver (sHRSF).

肿瘤组织中GPC3、Hepatocyte、CD_(34)检测对AFP阴性肝细胞癌的辅助诊断作用

目的评价肿瘤组织中磷脂酰肌醇蛋白聚糖3(GPC3)、Hepatocyte、CD34蛋白检测对甲胎蛋白(AFP)阴性肝细胞癌(HCC)的辅助诊断作用。方法回顾性分析48例癌细胞AFP阴性的HCC组织及其癌旁组织中GPC3、Hepatocyte、CD34蛋白表达特点。结果 48例HCC及其癌旁组织中GPC3蛋白阳性率分别为91.7%(44/48)、2.1%(1/48),两者比较P<0.01;Hepatocyte蛋白阳性率分别为95.8%(46/48)、100.0%(48/48),两者比较P>0.05;CD34蛋白阳性率分别为100.0%(48/48)、4.2%(2/48),两者比较P<0.01。HCC组织中GPC3与Hepatocyte表达呈正相关(r=0.314,P<0.05)。结论检测肿瘤组织中GPC3、Hepatocyte和CD34蛋白有助于AFP阴性原发性HCC的诊断。

Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues

AIM To study the changes of gene expression of hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFr) in hepatocellular carcinoma (HCC) tissue and nontumorous liver tissue and the relationship between these changes and the biological behavior of the tumor.METHODS Gene expression of HGF and HGFr in 26 cases of HCC tissue and their adjacent nontumorous liver tissues was determined with digoxigenin-labeled DNA probes.RESULTS Positive expression of HGF in HCC tissue was similar to that in the adjacent nontumorous liver tissue, but positive rate of HGF expression was lower than HGFr gene expression. However, HGFr expression was higher in the metastatic cases than in those without metastasis. It was found that HGFr was overexpressed in HCC tissue as well as in the adjacent nontumorous liver tissue.CONCLUSION There seems to be a close relationship between overexpression of HGFr gene and tumor metastasis, and the HGF and HGFr system plays an important role in regulating tumor growth and metastasis.

Protective Actions of Blumea Flavanones on Primary Cultured Hepatocytes and Liver Subcellular Organelle against Lipid Peroxidation

To search for the protective actions of blumea flavanones (BFs) on hepatocytes and hepatic subcellular organelle against lipid peroxidation, monkey′s hepatocytes were isolated and cultured with or without blumea flavanones, then damaged by FeSO 4 cysteine or CCl 4. The lipid peroxidation (malondialdehyde production) and alteration in hepatocyte membrane (leakage of GPT) were estimated. Hepatic subcellular organelles were also isolated and incubated with or without blumea flavanones, then injured by FeSO 4 ascorbate. The generation of malondialdehyde(MDA) was measured. It was found that BFs 10 and 100 μmol·L 1 inhibited the MDA generation and GPT (glutamic pyruvic transaminase) leakage out of hepatocytes that were induced by CCl 4 or FeSO 4 cysteine. BFs could prevent lipid peroxidation initiated by FeSO 4 ascorbate in subcellular organelle suspension. Among BFs, BF 2 possessed the strongest activity. Conclusion: Blumea flavanones possess antioxidation activities that protect monkey′s hepatocytes and hepatic subcellular organelle against injuries induced by FeSO4 or CCl 4.

Protective effects of polydatin against CCl_4-induced injury to primarily cultured rat hepatocytes

AIM To investigate the protective effects of polydatin (PD) against injury to primarily cultured rat hepatocytes induced by CCl 4. METHODS Rat hepatocytes were separated by methods of liver infusion in vivo and cultured medium (7 5×10 5 cells/mL). Two mL or 0 2mL was added into 24 well or 96 well plates respectively. Twenty four hours after cell preculture, PD at concentrations of 10 -7 mol/L-10 -4 mol/L was added into each plate. At the same time injury to hepatocytes was induced by adding 10mmol/L CCl 4. Then, 0 1mL or 1mL culture solution was removed from the 96 well or 24 well plates at 6h , 12h , 24h and 48h after CCl 14 intoxication respectively for the determination of GPT, GSH and MDA. At 48h , the survivability of rat hepatocytes was assayed by the MTT colormetric method. RESULTS After CCl 4 challenge, the release of GPT and the formation of MDA in rat hepatocytes markedly increased and maintained at a high level in 48h , whereas PD with different concentrations could markedly inhibit this elevation with 10 -5 mol/L PD having the strongest effects and inhibiting rate was over 50%. PD could also improve the decreased content of GSH caused by CCl 4 in accordance with the doses used. CCl 4 evidently decreased the hepatocyte survivability from 91 0%±7 9% to 35 4%±3 8%. On the other hand, PD at 10 -7 mol/L-10 -4 mol/L could reverse this change and improve the cell survival rates to 56 1%±5 2%, 65 8%±5 0%, 88 7%±6 8% and 75 2%±7 3%, respectively. CONCLUSION PD at 10 -7 mol/L-10 -4 mol/L could protect primarily cultured rat hepatocytes against CCl 4 induced injury.

Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.

Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination

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.

Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells,hepatocytes and fibroblasts in rats

AIM To study the mechanism of Fuzhenghuayu (FZHY) decoction on anti liver fibrosis. METHODS FZHY 10% decoction sera was incubated with rat normal subcultured hepatic stellate cells (HSC) and fibrotic primarily cultured HSC, normal and fibrotic hepatocytes and subcultured skin fibroblasts separately. Cell intracellular and extracellular collagen synthesis rates were measured by the method of Proline impulse and collagenase digestion. RESULTS For primarily cultured HSC and hepatocytes, both of intracellular and extracellular collagen synthesis rates decreased in the drug sera group. For the normal subcultured HSC and primarily cultured hepatocytes, the extracellular collagen secretion was decreased obviously by the drug sera, and intracellular collagen synthesis rates were inhibited to some extents. For fibroblasts, both intracellular and extracellular collagen synthesis rates were inhibited some what, but no significant differences were found. CONCLUSION The mechanism of FZHY decoction on anti liver fibrosis may be associated with inhibition of liver collagen production.

Assessing the anti-estrogenic activity of sodium pentachlorophenol in primary cultures of juvenile goldfish (Carassius auratus) hepatocytes using vitellogenin as a biomarker

Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.

Hepatocyte cryopreservation:Is it time to change the strategy?

Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative,or at least as a bridge,to orthotopic liver transplantation.The success of such a therapeutic approach remains limited by the quality of the transplanted cells.Cryopreservation remains the best option for long-term storage of hepatocytes,providing a permanent and sufficient cell supply.However, isolated adult hepatocytes are poorly resistant to such a process,with a significant alteration both at the morphological and functional levels.Hence,the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols,as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes.Intracellular ice formation or exposure to hyperosmotic solutionsremains the main phenomenon of cryopreservation process,and its effects on cell quality and cell death induction will be discussed.The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing.Such strategies,such as vitrification,will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies.

Hepatocyte nuclear factor 4-alpha involvement in liver and intestinal inflammatory networks

Hepatocyte nuclear factor 4-alpha (HNF4-&#x003b1;) is a nuclear receptor regulating metabolism, cell junctions, differentiation and proliferation in liver and intestinal epithelial cells. Mutations within the HNF4A gene are associated with human diseases such as maturity-onset diabetes of the young. Recently, HNF4A has also been described as a susceptibility gene for ulcerative colitis in genome-wide association studies. In addition, specific HNF4A genetic variants have been identified in pediatric cohorts of Crohn&#x02019;s disease. Results obtained from knockout mice supported that HNF4-&#x003b1; can protect the intestinal mucosae against inflammation. However, the exact molecular links behind HNF4-&#x003b1; and inflammatory bowel diseases remains elusive. In this review, we will summarize the current knowledge about the role of HNF4-&#x003b1; and its isoforms in inflammation. Specific nature of HNF4-&#x003b1; P1 and P2 classes of isoforms will be summarized. HNF4-&#x003b1; role as a hepatocyte mediator for cytokines relays during liver inflammation will be integrated based on documented examples of the literature. Conclusions that can be made from these earlier liver studies will serve as a basis to extrapolate correlations and divergences applicable to intestinal inflammation. Finally, potential functional roles for HNF4-&#x003b1; isoforms in protecting the intestinal mucosae from chronic and pathological inflammation will be presented.

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.