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Background: Hepatocyte nuclear factor 4α (HNF4α) plays an important role in the development of liver,and studies demonstrate that it is correlated with the pathogenesis of hepatocellular carcinoma (HCC).However,the regulatory effect of HNF4α on expression of vascular endothelial growth factor (VEGF) in human HCC cell lines and tube formation of human umbilical vein endothelial cell (HUVEC) is not yet clear.Aims: To investigate the effect of HNF4α on expression of VEGF in human HCC cell lines and tube formation of HUVEC.Methods: Lentiviral vector overexpressed HNF4α was constructed,and then transfected into HepG2 and SMMC-7721 cells (experimental group),cells transfected with lentiviral blank vector and cells without transfection were served as negative control group and blank control group,respectively.The mRNA and protein expressions of HNF4α,VEGF were detected by qRT-PCR and Western blotting,respectively.The conditioned media of HepG2 and SMMC-7721 cells were co-cultured with HUVEC,and number of HUVEC tube formation was measured.Results: HepG2 and SMMC-7721 cells with stable overexpression of HNF4α were successfully established.Compared with negative control group and blank control group,mRNA and protein expressions of VEGF in experimental group were significantly decreased (P＜0.05),and number of HUVEC tube formation was significantly decreased (P＜0.05).Conclusions: HNF4α can significantly inhibit the expression of VEGF in HepG2 and SMMC-7721 cells and tube formation of HUVEC.
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<p><b>OBJECTIVE</b>To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver.</p><p><b>METHODS</b>Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC).</p><p><b>RESULTS</b>The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not.</p><p><b>CONCLUSION</b>The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.</p>
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Humans , Albumins , Alginates , Capsules , Cell Cycle , Cell Survival , Cryopreservation , Dimethyl Sulfoxide , Hepatectomy , Hepatocytes , Cell Biology , Perfusion , PolylysineABSTRACT
Objective To observe the effects of hepatocyte-like cells differentiated from human umbilical cord mesenchymal stem cells (HuMSCs) on the liver function of the rats with liver cirrhosis.Methods Carbon tetrachloride was used to prepare rat model of liver cirrhosis.Then the rats in the experimental group received portal vein injection of 1 ml differentiated hepatocyte-like cells (1 × 107) ; the mesenchymal stem cell (MSC) group was injected with the same volume and number of MSCs; the model group was injected with the same volume of saline (NS) ; the normal rats were treated as control group.After transplantation,the rat angular vein blood and liver tissue were obtained for testing.Results One week after transplantation,compared with the model group,levels of serum alanine aminotransferase (ALT),aspartate aminotransferase (AST) and total bilirubin (TBil) in the experimental group significantly decreased (P ＜0.05),while the albumin (Alb) level increased significantly (P ＜0.05).Compared with the MSC group,the level of Alb in the experimental group also significantly increased (P ＜ 0.05),but there were no differences between the two groups of ALT,AST and TBil.4 weeks after transplantation,compared with the model group,levels of serum ALT,AST and TBil in the experimental group also significantly decreased (P ＜ 0.05),while Alb level increased significantly (P ＜ 0.05).Compared with the MSC group,the differences of the levels of Alb,ALT,AST and TBil were all statistically significant (P ＜ 0.05).Real-time PCR test results showed that the expressions of four liver-related genes of the MSC group and experimental group significantly increased comparing with the model group (P ＜ 0.05).And the experimental group showed higher expression level comparing with the MSC group (P ＜ 0.05).Conclusion The differentiated hepatocyte-like cells could improve hepatic function of patients with liver cirrhosis to a certain degree and showed greater advantage than MSC.
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Objective To investigate the effects of soluble components derived from bone marrow mesenchymal stem cells (BMSCs) on the expression of vascular endothelial growth factor (VEGF) and liver regeneration caused by 70％ portal branch ligation (PBL) in rats.Methods Isolated and cultured BMSCs were lysed by sonication.PBL was performed in male SD rats followed by splenic injection of BMSCs or PBS as control.Animals were analyzed for liver regeneration index,hepatocytes proliferation,hepatic function,histopathological changes,and hepatic genes expression.Expression of VEGF was assessed by Western blot and immunohistochemistry.Results The liver regeneration index increased in the BMSCs group especially 2 and 5 days after PBL compared with the control group (P＜0.05) and reached (51.71±1.62)％ and (76.82±0.81)％ respectively.A 2-fold increase was showed in the PCNA labeling index of hepatocytes in rats treated with BMSCs compared with the control group (P＜0.05).Histopathological findings showed that vacuolar change and sinusoidal congestion were lower in the BMSCs group.Alanine transaminase (ALT) and Aspartate transferase (AST) showed no significant difference between the two groups (P＞0.05).On post operation day 2,hepatic interleukin-6 (IL6),tumor necrosis factor α (TNFα),hepatocyte growth factor (HGF),vascular endothelial growth factor A (VEGFA),and vascular endothelial growth factor 2 (VEGFR2) mRNAs tended to increase in the BMSCs group (P＜0.05) while transforming growth factor β1 (TGFβ1) mRNA decreased (P＜0.05).Western blot showed that the expression level of VEGF in the two groups were equal 2 and 5 days after surgery (P＞0.05).On day 2 post operation,positive VEGF immunoreactivity was present in both pericentral and periportal hepatocytes in the BMSCs group,while only in periportal hepatocytes in the control group.Conclusion These results demonstrate that BMSCs accelerated liver regeneration caused by PBL,which may result from hepatoprotection,enhanced hepatocyte proliferation,and VEGF-mediated angiogenesis early after the operation,potentially creating a new avenue for the study of hepatic regeneration.
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Stem cells refer to undifferentiated or inad equately differentiated cells that have the ability to become a variety of tissues,regenerate organs,and self expand.There is strong evidence that stem cells have abilities of self renewal differentiation,immune regulation,and a potential for targeted therapy,which all together support development of stem cell transplantation.Stem cell transplantation thera py has become a hot topic in recent years,and the basic theory and clinical application have made great progress.The a bilities of stem cells make their transplantation ideal to pro mote liver regeneration,inhibit liver fibrosis,and provide treatment for many diseases.This article will review the cur rent status and outlook of stem cell transplantation.
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Remarkable progress has been achieved in the transplantation of mesenchymal stem cells (MSCs)for the treatment of liver injury and hepatic failure.However,there are obstacles such as low engraftment capacity,tumorigenesis,and a fibrogenic potential that all hamper the use of MSCs in clinical trials.Therefore,it is worthwhile to talk about the alternatives that will increase the safety and efficacy of MSCs therapy.To date,applications of MSCs-derived hepatocytes,genetically modified MSCs,or MSC-conditioned medium for promoting liver regeneration have shown encouraging results.This review summarizes the current applications of MSCs in the study of hepatic regeneration.
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Primary hepatocellular carcinoma progresses from liver fibrosis and cirrhosis to eventually result in liver failure and distant metastasis.Surgical resection is the preferred method of treatment for liver cancer while interventional treatment and liver transplantation are the choices to treat end-stage liver cancer.Unfortunately,partial hepatectomy and interventional treatment are not ideal due to the resulting consequence of hepatocyte dysfunction.Extensive clinical application of liver transplants is limited by the lack of available donors and high costs.Over the past decade,researches on bone marrow mesenchymal stem cells (BMSCs)have made remarkable achievements in the medical field.In this review,we summarize the recent progress of BMSCs in the treatment of liver diseases.
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Objective To investigate the mechanism by which epidermal growth factor (EGF) accelerates the growth of estrogen-negative breast cancer cell line MDA-MB-435S. Methods Under the effect of EGF cyclin D_1 was measured by Western blot and cell-cycle was assayed by FCM. Results EGF enhanced cyclin D_1 expression. Protein kinase C (PKC) inhibitor Go6976 blocked cyclin D_1 over-expression caused by EGF. In EGF group G_0/G_1 was 69.36%, phase S was 22.77% and PI was 0.31. Differences were all significant compared with control (P
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Objective To analyze the clinicopathologic factors related to the prognosis of gallbladder carcinoma.Methods A retrospective analysis was conducted on 61 cases of advanced gallbladder carcinoma who were operated on from 2001 to 2006 in Nanjing First Hospital.Results According to the 6th UICC/AJCC TNM classification and staging of gallbladder carcinoma,of the 61 patients,8 patients were stage IB,22 were IIA,11 were IIB,14 were III and 6 were IV.Palliative cholecystectomy and/or bile duct drainage procedures were performed in 24 patients;removal of gallbladder plus regional lymph nodes and fatty tissue dissections were performed in 14 cases;radical resection was performed in 7 patients;removal of gallbladder and part of liver in combination with resection of bile duct(radical operatian) was performed in 13 patients;1 patient had extensive radical operation including pancreaticoduodenectomy.In 37 patients,18 had positive lymph node metastasis.Fifty-two patients were followed up,41(67.2%) patients were alive at 6 months after operation,26(42.6%)were alive at 12 months,10(16.4%) were alive at 24 months,and 7 cases were alive at 36 months,1 patient is still alive 5 years after operation;51 patients died within 2 years after operation.The symptoms of recurrence in 31 patients included progressing jaundice(21 cases),metastasis in liver(14 cases),and refractory ascites(12 cases).Five patients died of non-bile duct disease.Conclusions The prognosis of gallbladder cancer has a close relationship with the clinical stage and method of resection.