Objective To investigate the effect of deoxyribonucleic acid (DNA) methylation on the expression of hepatocyte nuclear factor-4α (HNF4c) and its role in the expression of HNF4α regulated by transforming growth factor-β1 (TGF-β1).Methods The expression of HNF4αP1 mRNA in six human hepatoma cell lines (HepG2,Huh-7,Hep3B,SMMC-7721,BEL-7405 and FOCUS),20 hepatic carcinoma specimens and corresponding adjacent tissues was detected by real-time reverse transcription polymerse chain reaction (real-time RT-PCR).The methylation status of the promoter region of HNF4αP1 in six human hepatoma cell lines was examined by bisulfite sequencing PCR (BSP).FOCUS cells were treated with 5-aza-2'-deoxycytidine (5-AZA-CdR) and then the methylation status of the promoter region of HNF4αP1 was examined by BSP.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR.The six human hepatoma cell lines were treated with TGFβ1 and the expression of HNF4αP1 mRNA was detected by real-time RT-PCR.FOCUS cells were cotreated with 5-AZA-CdR,TGF-β1 and 5-AZA-CdR.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR,and t test was performed for statistical analysis.Results Among 20 human hepatic carcinoma specimens and corresponding adjacent tissues,the expression of HNF4αP1 mRNA of 13 human hepatic carcinoma specimens was lower than that of corresponding adjacent tissues (t=2.350,P＜0.05).The relative quantity of the expression of HNF4αP1 mRNA was higher in Hep3B,HepG2 and Huh-7 cells,whereas that in SMMC-7721,BEL-7405 and FOCUS cells was lower.The methylation of the promoter region of HNF4αP1 in HepG2,Huh-7 and Hep3B was lower,but that in SMMC-7721,BEL-7405 and FOCUS was higher.Along with the increasing of the concentration of 5-AZA-CdR (0,0.1,1.0 and 2.5 μmol/L),the degree of the methylation of the promoter region of HNF4αP1 in FOCUS cells gradually decreased (61％,46％,32％ and 27％),and however the relative quantity of the expression of HNF4αP1 mRNA gradually increased ((9.661 ± 0.336)×10-7,(2.001±0.432)×10-6,(3.689±0.714)×10-6and (4.732±2.451)×10-6).After stimulated with TGF-β1,the relative quantity of the expression of HNF4αP1 mRNA was downregulated in HepG2,Huh-7 and Hep3B cells in which the methylation of the promoter region was low (t=12.994,8.441,and 9.032,all P＜0.01).There was no significant difference in the relative quantity of the expression of HNF4αP1 mRNA in SMMC-7721,BEL-7405 and FOCUS cells in which the methylation of the promoter region was high (all P ＞ 0.05).The relative quantity of the expression of HNF4αP1 mRNA in 5-AZA-CdR treated FOCUS cells ((4.972±0.035) × 10-6) was higher than that of control group ((1.411 ± 0.104) × 10-6) and the difference was statistically significant (t=13.212,P＜0.01).The relative quantity of the expression of HNF4αP1 mRNA in FOCUS cells co-treated with 5-AZA-CdR and TGF-β1 was lower than that in cells treated with 5-AZA-CdR alone and the difference was statistically significant ((1.181 ± 0.132) × 10-6 vs (4.972 ± 0.035) × 10-6,t=13.873,P＜0.01).Conclusions The expression of HNF4αP1 is down-regulated in hepatic carcinoma tissues.DNA methylation may regulate the expression of HNF4αP1 in hepatoma cells.The methylation of HNF4αP1 promoter region inhibits the regulating function of TGF-β1 in the expression of HNF4αP1.

Objective To investigate the effect of recombinant human tumor necrosis factor (rhTNF) on telomerase activity in hepatoma cell line HepG2 cells and HepG1 6 cells. Methods TRAP ELISA method was used to determine the telomerase activity in HepG2 and HepG1 6 cells which were treated by different concentrations of rhTNF; plasmid which had been inserted 800 bp of the hTERT promoter was transiently transfected into HepG2 cells by Lipofect, and different concentrations of rhTNF were added into culture media 2 hours later, then the activity of the hTERT promoter was tested 48 hours after transfection. Results The telomerase activity of HepG2 was suppressed by rhTNF in a dose dependent manner. The expression of hTERT promoter was inhibited linearly with the dose of rhTNF within the range of 10～1 000 IU/ml. Conclusions Above results suggest that the anti tumor activity of rhTNF may attribute to its inhibitory effect on hTERT promoter expression.

Objective To investigate the effect of taurine on proliferation / apoptosis of rat hepatic stellate cells in culture and the possible mechanism involved. Methods Rat hepatic stellate cells were incubated with different concentration of taurine. Cell proliferation was assessed by MTT colorimetric assay, cell cycle and apoptosis were analysed by flow cytometry, apoptotic morphology was examined by vital staining of acridine orange, c-jun and c-fos expression was determined by immunocytochemistry and computer video text analysis system. Results Administration of 5-50 mmol/L taurine into culture medium had no toxicity to hepatic stellate cells, but could significantly inhibit hepatic stellate cell proliferation in dose-dependent manner, increase the number of cell in G 0/G 1 phase and decrease the numbers in S phase. Taurine could also markedly inhibit c-jun and c-fos expressions ( P

Objective To construct the replication deficient recombinant adenoviruses (AdHGF) of human hepatocyte growth factor (HGF) and green fluorescent protein (GFP) cDNA derived from CMV promoter using homologous recombination in bacteria provided by AdEasy system and to further investigate the effect of HGF on human fetal hepatocyte growth. Methods The HGF cDNA was obtained from the plasmid-pUCHGF by digestion, and the shuttle plasmid-pAdTrack-CMV-HGF in which the HGF cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was co-transformed into bacteria with backbone vector AdEasy-1 to obtain the recombinant adenoviral plasmid-pAdHGF by homologous recombination. After packed in 293 cells, the recombinant adenoviruses-AdHGF was generated. The expression of HGF in human fetal hepatocyte was detected by RT-PCR and the effect of AdHGF on the cell cycle of fetal hepatocyte was examined by flow cytometry. Results The recombinant plasmid pAdHGF was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the third day after packing of the linearized pAdHGF in 293 cells and 4?10 10 efu/ml titer of AdHGF was obtained by CsCl gradient purification. Three days after the human fetal hepatocytes were infected by the viruses, expression of HGF in hepatocytes increased significantly and most of the hepatocytes in G 0/G 1 stages was changed to that in S and G 2/M stages.Conclusions AdHGF can be simply and rapidly generated using AdEasy system. The infection of human fetal hepatocytes by AdHGF could result in the high expression of HGF and promote the cell regeneration. AdHGF may serve as a new tool for hepatocyte transplantation and gene therapy of hepatic fibrosis.

BACKGROUND:The patients are suffering from peritoneal adhesions that are caused after abdominal operation. As so far, there is stil no effective drug or method that can completely prevent peritoneal adhesions. Carboxymethyl chitosan is a biocompatible and biodegradable biomedical material with anti-adhesion effects, which is an ideal biomaterial for prevention of peritoneal adhesion theoreticaly.
OBJECTIVE:To investigate the novel anti-adhesion properties of carboxymethyl chitosan anti-adhesion solution on the prevention of postsurgical adhesion in vivo in a rat model.
METHODS:Fifty-six adult male Wistar rats were randomly divided into four groups: 0.9% normal saline solution (group A), hyaluronic acid gels (group B), medical chitosan gels (group C) and carboxymethyl chitosan anti-adhesion solution (group D). The model of postoperative intestinal adhesion was established by making cecal scratches/abdominal wal defects. Al the rats were scarified after 2 or 3 weeks. Whole blood was colected by cardio-puncture, lung tissue and tissue adhesion were stripped. The incidence and degree of adhesions, histological effects, expression of transforming growth factor-β1 (TGF-β1), the amounts of hydroxyproline and white blood cels were observed.
RESULTS AND CONCLUSION:The formation of postsurgical adhesions in groups B, C and D was significantly decreased, which was lighter than that of group A (P < 0.05). Furthermore, the adhesion formation in group D was significantly decreased in comparison with group A (P < 0.01). At the same time, the levels of transforming growth factor-β1, hydroxyproline and white blood cels in group D were lighter than those of group A (P < 0.05), and the histopathological results indicated that a marked reduction in inflammatory cels and fibroblasts. Carboxymethyl chitosan anti-adhesion solution can effectively reduce the degree and incidence of postoperative adhesion, and it is becoming a promising drug delivery system in the context of postsurgical anti-adhesion.

One of the most important characteristics of adult liver is its regenerative ability to maintain its original mass and function after injury. With remarkable self-replication ability, hepatocytes play a critical role in liver regeneration. When there is a restriction in the proliferation capacity and/ or a massive loss of mature hepatocytes,other cells in liver can contribute to liver regeneration in different ways. Deep study on the effects and mechanisms of various hepatic cells’contribution to liver regeneration is helpful for increasing the understanding about liver regeneration and providing new insights for diagnosis and management of various advanced liver diseases.

Objective To investigate the regulation effect of hepatocyte nuclear factor (HNF) 1α on the gene expression profile and the signal pathways in HuH7 cells.Methods The expression of HNF1α was increased or decreased in HuH7 cells by Lenti-virus carrying HNF1α or shHNF1α.The expression profile of the cells after treated was examined by microarray technology.The difference expressed gene regulated by HNF1α were screened and the pathway was analyzed with DAVID software and related analysis system.The regulation effect of HNF1α on transforming growth factor (TGF)β signal pathway was detected by reporter gene test and the regulation role of HNF1α on related genes of TGFβ signal pathway was determined by real-time polymerase chain reaction (PCR) and Western blotting assay.Results The expression of HNF1α in HuH7 cells was significantly up-regulated by Lenti-virus carrying HNF1α gene (Lenti-HNF1α) and which was down regulated by Lenti-virus with shHNF1α gene (LentishHNF1 α).Expression profile analysis revealed that 339 genes were positively up regulated two times by HNF1α and 325 genes were negatively down regulated two times.Signal pathway analysis revealed that HNF1α regulated drug metabolism,biosynthesis of unsaturated fatty acids and glycolysis/gluconeogenesis metabolism signal pathways.Moreover,it also involved in the regulation of TGFβ、nuclear factor (NF)-κB and p53 tumor-related signal pathways.Furthermore,Luciferase reportor gene experiment indicated that up-regulated HNF1α could inhibit the activation of TGFβ signal pathway.And the results of real-time PCR and Western blotting verified that up-regulated HNF1α could inhibit TGFβ signal pathway related gene c-myc and TGFβ1 and then inhibited the activation of TGFβ signal pathway.Conclusion HNF1α broadly affects the gene expression profile and the tumor genesis and development related signal pathways in HuH7 cells,furthermore,HNF1α can inhibit the activation of TGFβ signal pathway.

Objective To prepare proanthocyanidins/bletilla striata polysaccharide/chitosan microspheres ( PC/BSP/CTS ) and the physic-chemical characterizations were investigated.Methods The PC/BSP/CTS microspheres were prepared by spray drying method.The morphology of PC/BSP/CTS microspheres was observed by Scanning Electron Microscopy(SEM), and its physic-chemical characteristics such as diameters, release in vitro, moisture content, swelling ratio, solid density were studied.ResuIts The PC/BSP/CTS microspheres were successfully prepared by spray drying method, SEM showed that PC/BSP/CTS microspheres had the spherical shape with smooth surfaces.The diameters of microsphere A, B and C were 10~20, 2~15, 10~25μm.The in-vitro release showed that the cumulative release of three kinds of microspheres A, B, C was 25.07 %, 38.83 %and 60.00 % in 24 h, which had no burst release, while with time prolonged to 48 h, the cumulative release was 28.89%, 43.17% and 72.86%, respectively.The results of moisture content, swelling ratio, solid density were 15.35% ~23.51%, 46.50% ~105.80%, 0.375 ~0.496. ConcIusion The PC/BSP/CTS microspheres are successfully prepared by spray drying method which possess good characteristics and sustained-release effect, which would be as a good pulmonary drug delivery system.

The thymic cortex is the important site for thymocyte differentiation, maturation and selection. Direct cell-cell interactions between thymocytes and distinct stromal cells are an important pattern to demonstrate the essential role in T-cell development. In order to approach the mechanism of their interactions, ultrastructural cytochemical localization of 5'-nucleotidase activity in the thymic cortex of BALB/c mice were investigated by means of Robinson and Karnovsky method using 5'-AMP or 5'-IMP as the substrate, cerium as the capture agent, and levamisol (an inhibitor of nonspecific alkaline phosphatase) was also used. The cytochemical distribution of the reaction products from both the 5'-AMPase and 5'-IMPase activity was essentially similar. The enzyme activity was localized on the extracellular side of the plasma membrane of the epithelial reticular cells and some thymocytes, especially on their contact faces with each other. 5'-nucleotidase activity was also found in the lysosomes of macrophages and in the vacuoles, and vesicles near the plasma membrane and some lysosomes of epithelial reticular cells. The biological significance of the 5'-nucleotidase in thymic cortex was discussed.

Objective To prepare thermosensitive chitosan (CTS) hydrogels containing astragalus polysaccharides (APS)/CTS microshperes (MS), and evaluate its physicochemical properties. Methods The APS/CTS MS (APS-MS) were prepared by spray drying method, and characterized by Scanning Electron Microscopy (SEM) and Laser Granularity Analyzer. Depending on the gelation temperature and gelating time, thermosensitive CTS hydrogels (HG) containing APS-MS (APS-MS-HG) were optimized by signal factor experiments, and the morphological characteristics were observed by SEM. In vitro release behaviors of APS-MS, hydrogels containing APS (APS-HG) and APS-MS-HG in pH 6.8 phosphate buffer were evaluated by dialysis tube method. Results The APS-MS were well dispersed with nearly spherical shapes and slightly wrinkled surfaces. The surface weighted mean D[3,2] of APS-MS was 8.078μm. The optimal APS-MS-HG, APS-MS-HG J, contained 3.012% APS-MS which were agitated with a magnetic stirrer for 3h. Observed by SEM, APS-MS were stayed spherical and dispersed unevenly in HG J, but the porous structure of HG J was disappeared in APS-MS-HG J. The release of APS from APS-MS-HG J was without initial burst release, and the cumulative amount of APS was about 74.75% after 36h. Conclusion Suppressing the phenomenon of sudden release at the first stage of delivery, APS-MS-HG J holds great promise for topical applications as a sustained-release nasal delivery system.