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.

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.

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.

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.

OBJECTIVETo analyze the influence of optimal codon usage on the expression levels and immunogenicity of DNA vaccines, encoding the human papillomavirus type 6b (HPV 6b) E7 gene.

METHODSThe full length E7 gene of HPV 6b was modified to substitute human preferred codon for rarely used codon, and three mutations were introduced into the pRB binding site of HPV 6b E7 to eliminate its transformation potential. The codon optimized and mutated E7 gene (hu-mE7) were cloned into the Kpn I and EcoR I site of the pcDNA3 mammalian expression vector, the in vitro expression of the hu-mE7 gene and the immunogenicity of hu-mE7 DNA vaccine were compared with the wt-E7gene.

RESULTSThe in vitro expression of pcDNA3-hu-mE7 was much higher than the classical wt-E7 plasmid in monkey COS-1 cell line. Mice immunized intramuscularly with the pcDNA3-hu-mE7 showed that the codon modified E7 gene induced a stronger IFN-gamma ratios than the wt-E7 gene.

CONCLUSIONSThese results suggest that the optimized codon usage contributes to the enhancement of gene expression and immunogenicity of HPV 6b E7 gene.

Animals ; Cell Line ; Codon ; genetics ; Female ; Gene Expression ; Genes, Viral ; genetics ; Genetic Vectors ; Mice ; Papillomaviridae ; genetics ; Papillomavirus Vaccines ; Transfection ; Vaccines, DNA ; immunology ; Viral Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

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

Objective To investigate the current state of core competence of hospital clinical new nurses of different training mode so as to provide the basis for formulating targeted training mode . Methods Adopted order-form training mode that including internship and employment .Then using Competency Inventory For Registered Nurse to survey 104 new nurses who have registered in 2013 and analyzed the results . Results Total core capability score of new nurses was (158.40 ±25.95).And the dimensions score of critical thinking(27.81 ±3.72), clinical care (26.37 ±2.96), leadership ability (29.84 ±3.18), interpersonal relationships(26.05 ±5.47), legal/ethical practice(26.27 ±3.22), professional development (17.57 ±2.34) and education/consultation(20.43 ±3.14)among new nurses of order-form training was higher then that of non-order-form training nurses,and the differences between two groups showed statistically significance (t=6.207, 5.530,3.782,4.531,3.912,2.195,4.053,respectively;P<0.05).Conclusions Hospital should increase the proportion of order-form training, promote effective cooperation between institutions and hospitals and broaden the new ways of hospital talents construction .

Objective To discuss the correlation between the nurse′s humanistic care quality and the core competence of the low seniority nurses.Methods A total of 262 low seniority (work ≤3 years)registered nurses were selected from our hospital and completed the general information scale,nurse′s humanistic care quality scale and nurse′s core competence scale.Results The total score of humanistic caring quality of nurses was (1 1 0.41 ±1 1 .1 1 ).The total score of nurse′s core ability was (1 49.1 6 ±31 .08).Their total score and attributes were in the positive correlation (r =0.1 22-0.393,P <0.05).Conclusions Nurse′s humanistic care quality has the obvious correlation with the nurse′s core competence.When the hospital managers build up the humanistic care quality in the low seniority of nurses,it should carry on the care education,clear the care practicing and the standard of manage,improve the total level of the nurse′s humanistic care quality,training and develop the nurse′s core competence constantly in order to promote the total level nursing work.