Effects of cytokines on carbon tetrachloride-induced hepatic fibrogenesis in rats

AIM: To observe the possible effects of transforming growth factor (TGF) β1, interleukin (IL)-6, tumor-necrosis factor (TNF) α 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 (CCI4) (2 ml-kg^-1) twice a week. Fibrosisintervention group was given IL-10 at a dose of 4 μg-kg^-1 20 minutes before CCI4 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-β1,TNF-α, IL-6 and IL-10 were determined by enzyme-linkedimmunosorbent assay (ELISA). The liver tissues were taken for routine histological examination. RESULTS: Hepatic fibrosis was developed with the injection of CCI4. Values of the circulating TGFβ, TNFα, IL-6 and IL-10 in the control group were 25.495.56 ng.L^-1, 15.18±3.83ng.L^-1, 63.64±13.03 ng.L-^1 and 132.90±12.13 ng.L^-1,respectively. Their levels in the CCI4-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 TGFβ1, TNFα and IL-6 during hepatic fibrogenesis. These changes were pardally reversed by simultaneous administration of IL-10. The histological parameters, characterized by CCl4-intoxificatJon,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: TGFβ1, TNFα and IL-6 may play important roles during CCI4-induced hepatic fibrogenesis, and IL-10 may counterbalance their effects.

Overexpression of HBxAg in hepatocellular carcinoma and its relationship with Fas/FasL system

AIM: 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.METHODS: 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.RESULTS: 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 (P＞0.05). 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 μg@ L-1 835.36±407.33 μg@L-1 and 238.27±135.29 μg@L-1. The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36±9.61iμg@ L-1, 173.63±18.74 μg@L-1 and 121.96±7.83 μg@ L-1.Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P＜0.01). 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 (P＞0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues.CONCLUSION: 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.

Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat

The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tetregulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the TRAIL gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K_I increased, and the proportion of α-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.

Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system

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 chainreaction (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 13-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific.CONCLUSION: COXIII 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.

Effect of Gas Antisolvent on Conformation of Polystyrene in Tolene：Viscosity and Small—Angle X—ray Scattering Study

Synchrotron radiation small-angle X-ray scattering(SAXS) and the viscosity technique were used to investigate the effect of dissolved CO2 in toluene on the conformation of polystyrene(PS) in the solution.The viscosity of PS solution decreases faster with increasing antisolvent CO2 pressure than that of the solvent in the absence of the polymer.the intrinsic viscosity [η] calculated using the wellknown Huggins equation decreases with antisolvent pressure.It was found that the second virial coefficient A2 and the apparent mean-square radius of gyration<Rg^2>^1/2 decreases with pressure of antisolvent CO2.All these phenomena can be attributed to the shrink of PS chain in the course of adding the gas antisolvent because the intercation between the polymer and solvent becomes weaker.The values<Rg^2>^1/2 at different pressures obtained from SAXS data agree reasonably with those calculated from Flory theory using the viscosity data determined in this work.This implies that Flory theory,which has been used widely for the solutions of polymers in liquid solvents,is also applicable to the polymer solution with gas antisolvent.