BACKGROUND: Under the cellular physiological condition, 15.3% N-acetylated chitosan has poor solubility which limits cellular microencapsulation process.OBJECTIVE: This study was designed to prepare 50% N-acetylated chitosan with 15.3% N-acetylated chitosan and investigate its feasibility for preparing chitosan microcapsule used for hepatocyte embedding after in conjunction with methacrylic acid-hydroxyethyl methacrylate-methyl methacrylate (MAA-HEMA-MMA) copolymer under the cellular physiological condition.DESIGN, TIME AND SETTING: This study, a control observation experiment, was performed at the Central Laboratory, First Hospital Affiliated to Jinan University, Guagnzhou, Guangdong Province, China between January and October 2006.MATERIALS: 15.3% N-acetylated chitosan was provided by Sigma-Aldrich Company, Singapore. Hepatocytes were acquired from male Wistar rats, weighing 250-300g, by two-step collagenase digestion method.METHODS: Hepatocytes were microencapsulated using 50% N-acetylated chitosan and MAA-HEMA-MMA copolymer at 25℃ under aseptic condition.MAIN OUTCOME MEASURES: Microcapsule permeability was expressed with the diffusion degree of fluorescein isothiocyanate (FITC)-dextran in the empty microcapsule. The homeostasis of microencapsulated hepatocytes was measured by mechanical shearing-crush tests. The albumin-synthesizing capability of microencapsulated hepatocytes was determined by enzyme-labeled immunosorbent assay (ELISA). Urea-synthesizing capability was tested using kits (Sigma Diagnostics, USA) at the wavelength of 540 nm by colorimetric assay. Cytochrome P450 activity was determined using confocal laser microscopes and quantitated by the fluorescence intensity of products of 0-dealkylated 7-ethoxy resorufin, a substrate of cytochrome P450IA1. The plate-cultured hepatocytes were taken as controls.RESULTS: With increasing mass concentration of 50% N-acetylated chitosan, the diffusion degree of Mr 20000 and Mr 40000 FITC-dextran presented with a decreasing tendency, while the diffusion degree of Mr 70000 FITC-dextran was low and did not alter markedly. When the microencapsulated hepatocyte density was 2×109 L-1, the broken rate of microcapsule was only about 6% after 24 hours of shaking. Under the same condition of shaking, empty microcapsules were all broken after 4 hours of shaking. After 1 day of culture, approximately 30μmol/d urea could be synthesized among 1×106 bepatocytes that was markedly higher than the plate culture experimental result, 15μmol/d. After 7 days of culture, urea-synthesizing capability was close between in the microencapsulated hepatocytes and in the plate cultured hepatocytes. In the first 3 days of culture, 10μg/d albumin was acquired from 1×106 hepatocytes, and after 7 days of culture, only about 5μg/d albumin was obtained. The albumin level in the whole culture process was higher than plate culture results. After 1 day of culture, cytochrome P450 activity in the microencapsulated hepatocytes was higher approximately 8 times compared to plate culture results. After 1 week of culture, cytochrome P450 activity still retained at 50% of 1-day culture level.CONCLUSION: Microcapsule prepared by 50% N-acetylated chitosan under the physiological condition has good permeability and structural stability during the process of culture. Compared with in vivo surface culture test, degenerated chitosan microcapsule is better favorable to in vitro function of hepatocytes.

BACKGROUND:Bioartificial liver could partial y replace the major liver functions, including detoxification, synthesis, secretion and biotransformation. OBJECTIVE:To use bibliometric indexes to track study focuses on bioartificial liver, and to investigate the relationships among geographic origin, impact factors, and highly cited articles indexed in Web of Science. METHODS:A list of citation classics for bioartificial liver was generated by searching the database of Web of Science-Expanded using the terms“artificial liver support system”or artificial liver or“bioartificial liver”. The top 33 cited research articles which were cited more than 100 times were retrieved. RESULTS AND CONCLUSIONS:Of 4 144 articles published, the 33 top-cited articles were published between 1992 and 2010. The highest citations paper was published in 2002, with a total of 668 citations, mean cited 55.67 per year. The total citations of 33 articles were 6 094 times, with a mean of 12.64 citations per article. These top-cited papers came from 11 countries, of which 12 articles came from the United States. University of Rostock led the list of classics with five papers. Harvard University and Massachusetts General Hospital ranked the second with four papers each. The 33 top-cited articles were published in 18 journals, predominantly Annals of Surgery and Hepatology, fol owed by Artificial Organs and Biotechnology and Bioengineering. Our bibliometric analysis provides a historical perspective on the progress of bioartificial liver research. Articles originating from outstanding institutions of the United States and published in high-impact journals are most likely to be cited.

OBJECTIVETo explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism.

METHODSLevels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Marn-Whitney U test, and Pearson's correlation coefficient test.

RESULTSThe HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08 delta CT; P=0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%; P=0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity.

CONCLUSIONHBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; Epigenesis, Genetic ; Female ; Gene Expression ; Hep G2 Cells ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Male ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Trans-Activators ; pharmacology

Objective : To investigate the role of member septin family (SEPT12)in human spermatogenesis and its influence on sperm motility and sperm ultrastructure. Methods : Whole exome sequencing (WES) was performed on peripheral blood DNA extracted from 375 patients with asthenoteratozoospermia , and a patient with idiopathic infertility carrying compound heterozygous mutation of SEPT12 was screened out. Sanger sequencing was performed to verify the mutation , and co⁃segregation analysis was performed in the family. The morphological abnormalities of sperm were analyzed by hematoxylin⁃eosin (HE) staining and scanning electron microscopy (SEM) , and the ultrastructural defects of sperm were analyzed by transmission electron microscopy (TEM) . Then the effects of the mutation on the level and position of the protein and the changes of the location and level of the defect structure markers were analyzed by Western blot and immune⁃fluorescence (IF) . Results : The compound heterozygous mutations c.C332A (p. T111K) and c. 406_416 del TGCTCGTATTG (p. q136 VFS ∗39) in the SEPT12 gene were screened and identified in a patient with asthenoteratozoospermia. The mutations were verified by Sanger sequencing , which was consistent with the co⁃segregation genetic pattern of the family. The mutations resulted in loss of protein expression , decreased sperm motility and sperm morphological deformities , mainly including short tail , curly tail and irregular sperm head. The ultrastructure of sperm showed that the annulus between the mid⁃piece and the principlepiece was missing , the acrosome membrane of sperm head fell off and the nucleus contained vacuoles. In the midpiece of sperm flagella , the arrangement of mitochondrial sheath was disordered , most of flagella axoneme central pair was absent , microtubules doublet was missing or disordered , and some radical spoke was absent. By Western blot and IF , the marker proteins of related structural components were detected , and the results showed that the level of SEPT4 protein decreased , SEPT6 protein unchanged , acrosomal related proteins ACTL7A and ACROSIN protein missing , and the expression levels of mitochondrial and axoneme related proteins TOMM20 , SPAG6 and RSPH3 protein significantly decreased. Conclusion The deletion of SEPT12 protein caused by SEPT12 gene mutation leads to the deletion of the annulus between the mid⁃piece and the principle⁃piece , and the abnormal assembly of sperm acrosome , mitochondrial sheath and flagella.