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Background Emerging evidence has demonstrated that fecal microbiota transplantation (FMT) has a promising therapeutic effect on mice with experimental colitis and patients with ulcerative colitis (UC), although the mechanism of FMT is unclear. Aim To evaluate the protective effect of FMT on UC and clarify its potential dependence on the gut microbiota, through association analysis of gut microbiota with colon transcriptome in mice. Methods Dextran sodium sulfate (DSS)-induced experimental colitis was established and fecal microbiota was transplanted by gavage. Severity of colon inflammation was measured by body weight, disease activity index, colon length and histological score. Gut microbiota alteration was analyzed through 16S ribosomal ribonucleic acid sequencing. The differentially expressed genes (DEGs) in the colon were obtained by transcriptome sequencing. The activation status of colonic T lymphocytes in the lamina propria was evaluated by flow cytometry. Results Compared with the DSS group, the weight loss, colon length shortening and inflammation were significantly alleviated in the FMT group. The scores of disease activity index and colon histology decreased obviously after FMT. FMT restored the balance of gut microbiota, especially by upregulating the relative abundance of Lactobacillus and downregulating the relative abundance of Clostridium_sensu_stricto_1 and Turicibacter. In the transcriptomic analysis, 128 DEGs intersected after DSS treatment and FMT. Functional annotation analysis suggested that these DEGs were mainly involved in T-lymphocyte activation. In the DSS group, there was an increase in colonic T helper CD4+ and T cytotoxic CD8+ cells by flow cytometry. FMT selectively downregulated the ratio of colonic CD4+ and CD8+ T cells to maintain intestinal homeostasis. Furthermore, Clostri dium_sensu_stricto_1 was significantly related to inflammation-related genes including REG3G, CCL8 and IDO1. Conclusion FMT ameliorated DSS-induced colitis in mice via regulating the gut microbiota and T-cell modulation.

AIM: To investigate the relationship between Interleukin-8 (IL-8) and proliferation, adhesion, migration, invasion and chemosensitivity of gastric cancer (GC) cells. METHODS: The IL-8 cDNA was stably transfected into human GC cell line MKN-45 and selected IL-8-secreting transfectants. The expression of IL-8 in human GC cell line KATO-III was inhibited by RNA interference. The expressions of mRNA and protein of IL-8 in GC cells were detected by real-time reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay (ELISA). RESULTS: The overexpression of IL-8 resulted in an increased cell adhesion, migration and invasion, and a significant resistance to oxaliplatin in MKN-45 cells. Inhibition of IL-8 expression with small interfering RNA decreased the adhesion, migration and invasion functions and oxaliplatin resistance in KATO-III cells. IL-8 increased NF-κB and Akt activities and adhesion molecules ICAM-1, VCAM-1, and CD44 expression in GC cells. CONCLUSION: Overexpression of IL-8 promotes the adhesion, migration, invasion, and chemoresistance of GC cells, indicating that IL-8 is an important therapeutic target in GC.

To compare the outcomes of laparoscopic and open splenectomy and azygoportal devascularization for portal hypertension.From June 2006 to March 2009, laparoscopic splenectomy and azygoportal disconnection (LSD) were performed on 28 patients with cirrhosis, bleeding due to portal hypertension, and secondary hypersplenism. Success was achieved in 26 patients. Demographic, intraoperative, and postoperative variables of the patients were compared.Success of laparoscopic splenectomy and azygoportal disconnection was achieved in all but two patients (7.14%) who required open splenectomy and azygoportal devascularization (OSD). The operation time was significantly longer in patients undergoing LSD than in those undergoing OSD (235 +/- 36 min vs 178 +/- 47 min, P0.05). The estimated intraoperative blood loss was much more in patients receiving OSD than in those receiving LSD (420 +/- 50 mL vs 200 +/- 30 mL, P0.01). The proportion of patients undergoing laparoscopic and open splenectomy and azygoportal disconnection who received transfusion of packed red blood cells during or after the operation was 23.08% and 38.46%, respectively (P0.05). The time of first oral intake was faster in patients after LSD than in those after OSD (1.5 +/- 0.7 d vs 3.5 +/- 1.6 d, P0.05). The hospital stay of patients after LSD was shorter than that of patients after OSD (6.5 +/- 2.3 d vs 11.7 +/- 4.5 d, P0.05). The pain requiring medication was less severe in patients after LSD than in those after OSD (7.69% vs 73.08%, P0.001). The overall complication rate was lower in patients after LSD than in those after OSD (19.23% vs 42.31%, P0.05).Laparoscopic splenectomy and azygoportal disconnection are the feasible, effective, and safe surgical procedure, and are advantageous over minimally invasive surgery for bleeding portal hypertension and hypersplenism.

AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells. METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 mg/mL G418 and named HL-7702/HBV-encoded X protein (HBx) cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively. RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semi-quantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control. CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10).Hepatic fibrosis was induced by CCl(4) administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCl(4)-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7(th) and 11(th) wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with beta-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h.Compared to group N in the 7(th) wk, MMP-2 and TIMP-1 mRNA increased in group C (P = 0.001/0.001) and group I (P = 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P = 0.001/0.001). In the 11(th) wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7(th) week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P = 0.001), and increased in group C (P = 0.001) while decreased in group I (P = 0.042) compared with that in the 7(th) wk. Same results were found by immunocytochemistry.Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

To examine the expressions of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in rat fibrotic liver and in normal rat hepatic stellate cells, and to investigate the changes in their expressions in response to treatment with interleukin-10 (IL-10) and platelet-derived growth factor (PDGF).Rat models of CCl4-induced hepatic fibrosis were established and the liver tissues were sampled from the rats with or without IL-10 treatment, and also from the control rats. The expressions of MMP-2 and TIMP-1 in liver tissues were detected by S-P immunohistochemistry, and their expression intensities were evaluated in different groups. Hepatic stellate cells (HSCs) were isolated from normal rat and cultured in vitro prior to exposure to PDGF treatment or co-treatment with IL-10 and PDGF. MMP-2 and TIMP-1 levels were measured by semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR).CCl4- induced rat hepatic fibrosis models were successfully established. The positive expressions of MMP-2 and TIMP-1 increased obviously with the development of hepatic fibrosis, especially in untreated model group (84.0% and 92.0%, P0.01). The positive signals decreased significantly following IL-10 treatment (39.3% and 71.4%, P0.01 and P0.05) in a time-dependent manner. TIMP-1 mRNA in PDGF-treated group was significantly increased time-dependently in comparison with that of the control group, but PDGF did not obviously affect MMP-2 expression. No difference was noted in TIMP-1 and MMP-2 expressions in HSCs after IL-10 and PDGF treatment (P0.05).MMP-2 and TIMP-1 expressions increase in liver tissues with the development of fibrosis, which can be inhibited by exogenous IL-10 inhibitor. PDGF induces the up-regulation of TIMP-1 but not MMP-2 in the HSCs. IL-10 inhibits TIMP-1 and MMP-2 expressions in HSCs induced by PDGF.

To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702.The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times.RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope, but not in HL-7702/pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression.HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.

To study the expression and serum level of HBxAg, Fas and FasL in tissues of HCC patients, and to assess the relationship between HBxAg and Fas/FasL system.Tissues from 50 patients with HCC were tested for the expression of HBxAg, Fas and FasL by S-P immunohistochemistry. Serum levels of sFas/sFasL and HBsAg/HBeAg were measured by ELISA assay. HBV X gene was detected by PCR in serum and confirmed by automatic sequencing. Fifty cases of liver cirrhosis and 30 normal controls were involved in serum analysis.The expression of HBxAg, Fas and FasL in carcinoma tissues was 96 %, 84 % and 98 %, respectively. Staining of HBxAg, Fas and FasL was observed predominately in cytoplasms, no significant difference was found in intensity between HBxAg, Fas and FasL (P0.05). HBxAg, Fas and FasL might express in the same area of carcinoma tissues and this co-expression could be found in most patients with HCC. The mean levels of sFas in serum from HCC, cirrhosis and normal controls were 762.29 +/- 391.56 microg.L(-1), 835.36 +/- 407.33 microg.L(-1) and 238.27 +/- 135.29 microg.L(-1). The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36 +/- 9.61 microg.L(-1), 173.63 +/- 18.74 microg.L(-1) and 121.96 +/- 7.83 microg.L(-1). Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P0.01). Serum HBV X gene was found in 32% of HCC patients and 46% of cirrhotic patients. There was no significant relationship between serum level of sFas/sFasL and serum X gene detection (P0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues.Our data suggest that HBxAg and Fas/FasL system plays an important role in the development of human HCC. Expression of HBxAg can leads to expression of Fas/FasL system which and reverse apoptosis of hepatocellular carcinoma induced by FasL.

AIM To compare KAI1 in cancer of papilla of Vater and pancreas to evaluate whether there are differences in biologic behavior which might account for prognosis. METHODS We compared the expression in 24 papillay and 29 pancreatic cancers using Northern blot analysis, immunochemical assay and in situ hybridization, and investigated whether early diagnosis or molecular differences predict the outcome in these tumor entities. RESULTS By Northern blot analysis there is no statistical difference of KAI1 levels in normal and cancerous papilla. No association between KAI1 mRNA expression and tumor stage or tumor differentiation was found in the tumors. By immunohistochemical assay, KAI1 staining in cytoplasm of papillary cancer cells was similar to that of normal papillary cells. By in situ hybridization, the results of KAI1 mRNA expression in normal and cancerous papilla were similar to those with immunohistochemical assay. The normal and cancerous pancreas tissues were also analyzed by the methods used in papillary samples. CONCLUSION Although the biologic roles of KAI1 have not been clarified, our results suggest that KAI1 may restrict the progression of malignant papillary cancer, but its expression might not have any effect on the characteristics of papillary tumor, whereas by the analysis of KAI1 gene, its reduced expression is closely related to the progression and metastases of pancreatic cancer.

AIM:To assess the diagnostic values of tumor markers for pancreatic cancer.METHODS:Pancreatic cancer-associated antigen from colonic mucosa (PCAAc), pancreas-specific antigen (PaA), pancreatic oncofetal antigen (POA) and minimolecular pancreatic antigen (mPOA) were detected by double antibodies Sandwich ELISA; CA19-9,elastase 1 (E1), human pancreatic elastase 1 (HPE1) and carcinoembryonic antigen (CEA) by radioimmunoassay (RIA); general activities of ribo-nuclease (RNase) and its isoenzymes (RNase I and RNase I) by biochemistry and PAEG;glycylproline dipeptidyl aminopeptidase (GPDA) by biochemistry andalpha 1-antitrypsin (alpha1AT) by rocket immunoelectrophoresis (rocket-IE).RESULTS:The detection of serum POA,mPOA,PaA,PCAAc,CA19-9,RNase and RNase I was able to differentiate pancreatic cancer from the benign disorders and non-pancreatic malignancies with a sensitivity from 66.75% to 80.0% and a specificity from 88.5% to 96.69%. POA, mPOA, PCAAc, HPE1, E1 and GPDA were related to the pancreatic cancer at the head which demonstrated higher sensitivity from 63.64% to 85.71%. The detection of serum HPE1 was especially helpful for the diagnosis of pancreatic cancer with smaller diameters. The determination of 3 or 4 kinds of tumor markers simultaneously would increase the detection rate of pancreatic cancer, which will be an important procedure for the diagnosis of this malignancy.CONCLUSION:A single test of tumor markers is helpful to detect pancreatic cancer clinically,but the determination of 3 or 4 kinds of tumor markers simultaneously would significantly increase the detection rate of pancreatic cancer, which will be an important procedure for the diagnosis of this malignancy.

AIM: To describe a population of outpatients in China infected by hepatitis B virus (HBV) and/or hepatitis C virus (HCV), and assess their current management status. METHODS: A multicenter, cross-sectional study of HBV- and/or HCV-infected patients was conducted from August to November, 2011 in western China. Patients ≥ 18 years of age with HBV and/or HCV infections who visited outpatient departments at 10 hospitals were evaluated, whether treated or not. Data were collected on the day of visit from medical records and patient interviews. RESULTS: A total 4010 outpatients were analyzed, including 2562 HBV-infected and 1406 HCV-infected and 42 HBV/HCV co-infected patients. The median duration of documented infection was 7.5 years in HBV-infected and 1.8 years in HCV-infected patients. Cirrhosis was the most frequent hepatic complication (12.2%), appearing in one-third of patients within 3 years prior to or at diagnosis. The HCV genotype was determined in only 10% of HCV-infected patients. Biopsy data were only available for 54 patients (1.3%). Antiviral medications had been received by 58.2% of patients with HBV infection and 66.6% with HCV infection. Nucleos(t)ide analogs were the major antiviral medications prescribed for HBV-infected patients (most commonly adefovir dipivoxil and lamivudine). Ribavirin + pegylated interferon was prescribed for two-thirds of HCV-infected patients. In the previous 12 mo, around one-fifth patients had been hospitalized due to HBV or HCV infection. CONCLUSION: This observational, real-life study has identified some gaps between clinical practice and guideline recommendations in China. To achieve better health outcomes, several improvements, such as disease monitoring and optimizing antiviral regimens, should be made to improve disease management.

To compare the effectiveness of standard triple, bismuth pectin quadruple and sequential therapies for Helicobacter pylori (H. pylori) eradication in a randomized, double-blinded, comparative clinical trial in China.A total of 215 H. pylori-positive patients were enrolled in the study and randomly allocated into three groups: group A (n = 72) received a 10-d bismuth pectin quadruple therapy (20 mg rabeprazole bid, 1000 mg amoxicillin bid, 100 mg bismuth pectin qid, and 500 mg levofloxacin qd); group B (n = 72) received the sequential therapy (20 mg omeprazole bid, 1000 mg amoxicillin bid, in 5 d, followed by 20 mg omeprazole bid, 500 mg tinidazole bid, 500 mg clarithromycin bid, for another 5 d); group C (n = 71) received a standard 1-wk triple therapy (20 mg omeprazole bid, 1000 mg amoxicillin bid, 500 mg clarithromycin bid). After all these treatments, 20 mg omeprazole bid was administrated for 3 wk. H. pylori status was assessed by histology, 13C-urea breath test and rapid urease test at baseline and 4-6 wk after completion of treatment. Ulcer cicatrization was assessed by gastroscopy. chi(2) test (P0.05) was used to compare the eradication rates and ulcer cicatrisation rates among the three groups.The eradication rate was 83.33% (60/72) in group A, 88.89% (64/72) in group B, and 80.56% (58/71) in group C. The ulcer cicatrisation rate was 86.44% (51/59) in group A, 90.16% (55/61) in group B, and 84.91% (45/53) in group C. The sequential therapy yielded a higher eradication rate and ulcer cicatrisation rate than the standard triple and bismuth pectin quadruple therapies. Statistically, the eradication rate of group B was significantly different from groups A and C (P0.05), but the difference of ulcer cicatrisation rate and side effects was not statistically significant among the three groups (P0.05). The three protocols were generally well tolerated.The sequential therapy has achieved a significantly higher eradication rate, and is a more suitable first-line alternative protocol for anti-H. pylori infection compared with the standard triple and bismuth pectin quadruple therapies.

To study the effects of interleukin-10 (IL-10) on the expression of alpha-smooth muscle actin (alpha-SMA), nuclear factor- kappa B(NF- kappa B) and Fas/Fas ligand (FasL) in hepatic stellate cells of experimental rats with hepatic fibrosis.Sixty clean SD rats were randomly divided into control group (group N), liver fibrotic group (group C) and IL-10 treatment group (group I). Control group received intraperitoneal injection of saline (2 mL/kg), twice a week. Fibrotic group was injected intraperitoneally with 50% carbon tetrachloride (CCl(4)) (2 mL/kg), twice a week. IL-10 treatment group was given IL-10 at a dose of 4 microg/kg 20 minutes before CCl(4) administration from the third week. Hepatic stellate cells (HSCs) were isolated from these rats at the seventh and eleventh weeks during the course of liver fibrosis, respectively. The expression of alpha-SMA and NF- kappa B in HSCs was measured by S-P immunohistochemistry. The expression of Fas and FasL mRNA was measured by RT-PCR. Furthermore, liver tissues were harvested from three groups at the same time.The CCl(4)- induced experimental rat hepatic fibrosis model was established successfully. The purity of extracted hepatic stellate cells was about 95% and the yield of hepatic stellate cells was 1.2-2.3 x 10(6)/g liver tissue averagely. The positive expression of alpha-SMA and NF- kappa B was 36.5% and 28.5% respectively in group N. The positive levels of alpha-SMA and NF- kappa B were increased significantly in group C compared to group N (P0.01). The positive signals decreased significantly (P0.05) in group I. In the 11th week, the HSCs of group I became round with visible pyknotic nuclei. The expression of NF- kappa B in group C was significantly increased in a time-dependent manner (P0.01), but there was no difference in the alpha-SMA expression (P0.05). The mRNA of Fas and FasL in group C was significantly increased in a time-dependent manner compared to that in control group. After treated with IL-10, the expression level of Fas and FasL was higher in group I than in group C.The positive expression of alpha-SMA and NF- kappa B in hepatic stellate cells is decreased by ectogenic IL-10 in liver fibrosis induced by CCl(4). The expression of Fas and FasL is increased in the course of liver fibrosis, and is further increased by IL-10. IL-10 could inhibit the activation of HSCs and cause apoptosis of activated HSCs.

Interleukin (IL)-10 is an important immunoregulatory cytokine produced by many cell populations. Numerous investigations suggest that IL-10 plays a major role in chronic liver diseases. IL-10 gene polymorphisms are possibly associated with liver disease susceptibility or severity. Recombinant human IL-10 has been produced and is currently tested in clinical trials. These trials may give new insights into the immunobiology of IL-10 and suggest that the IL-10/IL-10 receptor system may become a new therapeutic target.

Aim To study the effect of IL-10 on the expression of growth factors--transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. Methods Hepatic fibrosis was induced by CCl(4) administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. Results Rat hepatic fibrosis was developed with the increase of injection frequency of CCl(4), and HSCs were successfully isolated. At the 7th and 11th wk, TGF-beta1, EGF, and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P = 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-beta1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P = 0.005) and 11th wk (P = 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-beta1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-beta1 and EGF were paralleled with the above findings. Conclusion The expression of TGF-beta1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2 (0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P

To investigate the effects of platelet-derived growth factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells.Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group.The expression levels of Fas between the 4 groups had no significant differences (P0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091+/-0.007 vs 0.385+/-0.051, P0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126+/-0.008 vs 0.210+/-0.024, P0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513+/-0.016 vs 0.400+/-0.022, P0.01). No significant difference was found between group I and group P (P0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449+/-0.028 vs 0.513+/-0.016, P0.05).PDGF may promote proliferation of HSCs but is neutral with respect to HSC apoptosis. IL-10 may promote the apoptosis of HSCs by up-regulating the expressions of FasL and Bax and down-regulating the expression of Bcl-2, which may be involved in its antifibrosis mechanism.

AIM: To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC). METHODS: With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with β-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was peformed to confirm the binding of putative proteins to X protein in the yeast cells. RESULTS: Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through β-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (cox III). Furthermore, mating experiment identified that the binding of cox III to X protein was specific. CONCLUSION: cox III protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein.

Aim To observe the possible effects of transforming growth factor (TGF) beta(1), interleukin (IL)-6, tumor-necrosis factor (TNF) alpha and IL-10 on experimental rat hepatic fibrosis. Methods One hundred SD rats were divided randomly into the three groups. Control group received intraperitoneal injection of saline (2 ml/kg(-1)), twice a week. Fibrogenesis group was injected intraperitoneally with 50% carbon tetrachloride (CCl(4)) (2 ml/kg(-1)) twice a week. Fibrosis-intervention group was given IL-10 at a dose of 4 microg/kg(-1) 20 minutes before CCl(4) administration from the third week. At the fifth, seventh, and ninth weeks, 7 to 10 rats in each group were sacrificed to collect serum. Levels of TGF-beta(1), TNF-alpha, IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA). The liver tissues were taken for routine histological examination. Results Hepatic fibrosis was developed with the injection of CCl(4). Values of the circulating TGFbeta(1), TNFalpha, IL-6 and IL-10 in the control group were 25.49+/-5.56 ng/L(-1), 15.18+/-3.83 ng/L(-1), 63.64+/-13.03 ng/L(-1) and 132.90+/-12.13 ng/L(-1), respectively. Their levels in the CCl(4)-intoxication group were 31.13+/-6.41 ng/L(-1), 18.91+/-5.31 ng/L(-1), 89.08+/-25.39 ng/L(-1) and 57.63+/-18.88 ng/L(-1), respectively, and those in the IL-10-intervention group were 26.11+/-5.32 ng/L(-1), 13.99+/-1.86 ng/L(-1), 74.71+/-21.15 ng/L(-1) and 88.19+/-20.81 ng/L(-1), respectively. A gradual increase was observed in the levels of TGFbeta(1), TNFalpha and IL-6 during hepatic fibrogenesis. These changes were partially reversed by simultaneous administration of IL-10. The histological parameters, characterized by CCl(4)-intoxification, also seemed to be improved with IL-10 treatment, the collagen production was reduced at the ninth week and the histological activity index was decreased from 7.9+/-1.2 to 4.7+/-0.9. Conclusion TGFbeta(1), TNFalpha and IL-6 may play important roles during CCl(4)-induced hepatic fibrogenesis, and IL-10 may counterbalance their effects.

AIM: To evaluate the potential role of Nimesulide, a selective COX-2 inhibitor, in proliferation and apoptosis of gastric adenocarcinoma cells SGC-7901. METHODS: Cell counts and MTT assay were used to quantify the influence of Nimesulide in the proliferation of SGC-7901 cells. Transmission electron microscopy and flow cytometry were used to observe the induction of Nimesulide the apoptosis of SGC-7901 cells and influence in the distribution of cell cycle. The expression of P27kip1 protein was observed by immunocytochemical staining. RESULTS: SGC-7901 Cells treated with Nimesulide at various concentrations exhibited a profound dose- and time-dependent reduction in the proliferation rate over the 72 h test period. The highest survival rate of the cells was 78.7%, but the lowest being 22.7%. Nimesulide induced apoptosis of the cells in a dose-dependent and non-linear manner and increased the proportion of cells in the G0/G1 phase and decreased the proportion in the S and G2/M phase of the cell cycle. Meanwhile, Nimesulide could up-regulate the expression of P27kip1 protein. CONCLUSION: The induction of apoptosis and cell cycle arrest are both anti-proliferative responses that likely contribute to the antineoplastic action of nimesulide on SGC-7901 cells. The up-regulation of P27kip1 gene may contribute to the accumulation of these cells in the G0/G1 phase following treatment with Nimesulide. Selective COX-2 inhibitor may be a new channel of the chemoprevention and chemotherapy for gastric carcinoma.

AIM: To seek the X associated protein (XAP) with the constructed bait vector pAS2-1X from normal human liver cDNA library. METHODS: The X region of the HBV gene was amplied by PCR and cloned into the eukaryotic expression vector pAS2-1.The reconstituted plasmid pAS2-1X was transformed into the yeast cells and the expression of X protein (pX) was confirmed by Western blot analysis. Yeast cells were cotransformed with pAS2-1X and the normal human liver cDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with the LacZ report gene. Furthermore, segregation analysis and mating experiment were performed to eliminate the false positive and the true positive clones were selected for PCR and sequencing. RESULTS: Reconstituted plasmid pAS2-1X including the anticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed that reconstituted plasmid pAS2-1X expressed BD:X fusion protein in yeast cells. Of 5 × 106 transformed colonies screened, 65 grew in the selective SC/-trp-leu-his-ade medium, 5 scored positive for β-gal activity, and only 2 remaining clones passed through the segregation analysis and mating experiment. Sequence analysis identified that two clones contained similar cDNA fragment: GAACTTGCG. CONCLUSION: The short peptide (glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normal human liver cDNA library has difficulties in expressing the integrated XAP on yeast cells.