Hepatocyte is the core raw materials of bioartificial liver support system, primary hepatocyte is lim-ited to application because of short survival and difficult cul-ture in vitro. Porcine hepatocyte which has been used re-cently exist the risks of endogenous retrovirus transmission.With the development of molecular biology, it has been pos-sible that hepatocyte is immortalized recently. Immortalized hepatocytes have greatly significant to drug toxicology, bio-artificial liver support system and tissue engineering of liver.Therefore, we will review the prospects for research and ap-plication of immortalized hepatocytes.

Objective To investigate the clinical value of the precision liver surgery in the liver graft procurement for pediatric living donor liver transplantation.Methods The clinical data of 58 living donors of left hepatic lobe graft who were admitted to the Renji Hospital of Shanghai Jiaotong University from December 2012 to January 2014 were retrospectively analyzed retrospectively from December 2012 to January 2014.All the donors donated voluntarily and gratuitously and were approved by the ethics committee of the hospital.All the donors received computed tomography (CT),and the two dimensional data were converted to three dimensional images for evaluating the intrahepatic bile ducts and blood vessles,and the typs of the left hepatic arteries and veins were determined.The donor's liver graft volume was assessed by CT before operation.The standard liver volume of the donors and the recipients,and the volume of liver to be reseeted and the total liver volume were measured.A virtual surgery was conducted for designing the actual surgery.The liver graft was resected with the precision liver surgery technique.Patients were followed up by the out-patient examination and phone call till April 2014.Results The results of CT angiography confirmed that 28 donors were with type Ⅰ left hepatic artery,10 with type Ⅱ left hepatic artery and 20 with type Ⅲ left hepatic artery; 35 patients were with type Ⅰ left hepatic vein and 23 with type Ⅱ left hepatic vein.The left-lobe volume estimated by CT was (243 ± 65) mL.Liver graft procurement was successfully carried out on the 58 donors,including 7 left hemihepatectomy and 51 left lateral lobectomy.Two donors received cholecystectomy concomitantly.The actual volume of liver resected was (255 ±59) mL,and the error rate of the liver volume to be resected was 4.94％.The weight of the liver graft to the body weight of the recipient was 3.3％ ± 1.0％.The operation time and the volume of blood loss were (260 ± 89) minutes and (181 ± 35)mL,respectively.One donor received red blood cell infusion of 2 U.The time for gastrointestinal function recovery was (2.0 ± 1.1) days,and the time of drainage tube pull-off was (3.0 ± 1.2) days.The duration of postoperative stay was (7 ± 3) days.The white blood cells,hemoglobin,alanine transaminase,aspartate transaminase,total bilirubin,direct bilirubin and albumin were at the normal levels at the discharge.Two donors were complicated by incisional bleeding and fat liquefaction,and they were cured by symptomatic treatment.All the donors were followed up for a median time of 8.7 months.The donors were recovered well without complications during the follow-up.Conclusions Liver graft procurement guided by precision liver surgery has the advantages of high accurate rate,little injury to the liver of the donors,few postoperative complications and quick recovery of the donors.

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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.

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.

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.

OBJECTIVETo 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.

METHODSAdult 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).

RESULTSThe 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.

CONCLUSIONThe 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.

Albumins ; Alginates ; Capsules ; Cell Cycle ; Cell Survival ; Cryopreservation ; Dimethyl Sulfoxide ; Hepatectomy ; Hepatocytes ; cytology ; Humans ; Perfusion ; Polylysine ; analogs & derivatives