Effects of hepatitis E virus infection on interferon production via ISG15.

AIM: To assess the effects of hepatitis E virus (HEV) on the production of type I interferons (IFNs) and determine the underlying mechanisms. METHODS: We measured the production of interferon (IFN)-alpha and -beta (-α/ß) in genotype 3 HEV-infected C3A cells at different time points (0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay (ELISA). The expression levels of IFN-stimulated gene (ISG)15 in HEV-infected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3 (ORF3) or control plasmids (green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/ß and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/ß was also measured by ELISA. RESULTS: We showed that genotype 3 HEV could enhance the production of IFN-α/ß and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/ß and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/ß. Overexpression of ISG15 resulted in the reduction of IFN-α/ß. CONCLUSION: HEV may promote production of IFN-α/ß and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/ß.

Hepatitis B virus upregulates host expression of α-1,2-mannosidases via the PPARα pathway.

AIM: To assess the effects of hepatitis B virus (HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms. METHODS: We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG2.2.15, HepN10, HepAD38 and HepG2 by Western blot. Viral antigens (HBsAg and HBeAg) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV (PTT22-HBx, PTT22-HBs, PTT22-preS2, PTT22-preS1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids (PTT22-vector) were transfected into HepG2 cells. MK886 (PPARα) and GW9662 (PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked. RESULTS: We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsAg in all the cell lines. Levels of α-1,2-mannosidase in non-tumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBV-related HCC patients. Moreover, transfecting HepG2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662. CONCLUSION: Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.

A novel platform designed by Au core/inorganic shell structure conjugated onto MTX/LDH for chemo-photothermal therapy.

A novel platform making up of methotrexate intercalated layered double hydroxide (MTX/LDH) hybrid doped with gold nanoparticles (NPs) may have great potential both in chemo-photothermal therapy and the simultaneous drug delivery. In this paper, a promising platform of Au@PDDA-MTX/LDH was developed for anti-tumor drug delivery and synergistic therapy. Firstly, Au NPs were coated using Layer-by-Layer (LbL) technology by alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and MTX molecules, and then the resulting core-shell structures (named as Au@PDDA-MTX) were directly conjugated onto the surface of MTX/LDH hybrid by electrostatic attraction to afford Au@PDDA-MTX/LDH NPs. Here MTX was used as both the agent for surface modification and the anti-tumor drug for chemotherapy. The platform of Au@PDDA-MTX/LDH NPs not only had a high drug-loading capacity, but also showed excellent colloidal stability and interesting pH-responsive release profile. In vitro drug release studies demonstrated that MTX released from Au@PDDA-MTX/LDH was relatively slow under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. Furthermore, the combined treatment of cancer cells by using Au@PDDA-MTX/LDH for synergistic hyperthermia ablation and chemotherapy was demonstrated to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of the platform for cancer therapy.

Methotrexate intercalated layered double hydroxides with the mediation of surfactants: Mechanism exploration and bioassay study.

Methotrexatum intercalated layered double hydroxides (MTX/LDHs) hybrids were synthesized by the co-precipitation method and three kinds of nonionic surfactants with different hydrocarbon chain lengths were used. The resulting hybrids were then characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). XRD and FTIR investigations manifest the successful intercalation of MTX anions into the interlayer of LDHs. TEM graphs indicate that the morphology of the hybrids changes with the variation of the chain length of the surfactants, i.e., the particles synthesized using polyethylene glycol (PEG-7) present regular disc morphology with good monodispersity, while samples with the mediation of alkyl polyglycoside (APG-14) are heavily aggregated and samples with the addition of polyvinylpyrrolidone (PVP-10) exhibit irregular branches. Furthermore, the release and bioassay experiments show that monodisperse MTX/LDHs present good controlled-release and are more efficient in the suppression of the tumor cells.

Facile synthesis of methotrexate intercalated layered double hydroxides: particle control, structure and bioassay explore.

To study the influence of particle size on drug efficacy and other properties, a series of methotrexate intercalated layered double hydroxides (MTX/LDHs) were synthesized through the traditional coprecipitation method, using a mixture of water and polyethylene glycol (PEG-400) as the solvent. To adjust the particle size of MTX/LDHs, the dropping way, the volume ratio of water to PEG-400 and different hydrothermal treatment time changed accordingly, and the results indicate that the particle size can be controlled between 90 and 140 nm. Elemental C/H/N and inductive coupled plasma (ICP) analysis indicated that different synthesis conditions almost have no effect on the compositions of the nanohybrids. X-ray diffraction (XRD) patterns manifested the successful intercalation of MTX anions into the LDH interlayers, and it's also found out that different volume ratios of water to PEG-400 and variable dropping way can affect the crystallinity of the final samples, i.e., the volume ratio of 3:1 and pH decreasing are proved to be optimum conditions. Furthermore, both antiparallel monolayer and bilayers adopting different orientations are suggested for four samples from XRD results. Fourier transform infrared spectroscopy (FTIR) investigations proved the coexistence of CO3(2-) and MTX anions in the interlayer of the nanohybrids. MTX/LDH particles exhibited hexagonal platelet morphology with round corner and different dropping ways can affect the morphology greatly. Moreover, a DSC study indicated that longer time treatment can weaken the bond between the MTX anions and LDH layers. The kinetic release profiles told us that larger MTX/LDH particles have enhanced the ability of LDH layers to protect interlayer molecules. At last, the bioassay study indicated that the nanohybrids with larger diameters have higher tumor suppression efficiency.

MTX/LDHs hybrids synthesized from reverse microemulsions: particle control and bioassay study.

Reverse microemulsions have been used to control the growth of methotrexatum intercalated layered double hydroxides (MTX/LDHs) hybrids, and the influence of reaction temperature, water content (noted as ω) and MTX content (noted as R) on the properties of MTX/LDHs was systematically investigated. The synthesized hybrids were then characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and atomic force microscopy (AFM), etc. XRD and FTIR investigations manifest the successful intercalation of MTX anions into the interlayer of LDHs. The process of particle control has been explored emphatically, and it was found that temperature, water content, and addition of solutes can determine the structural evolution as well as the size of the "water pools" in the reverse microemulsions, while ω plays a critical role in the particle growth. Then in vitro release tests of all hybrids in pH 7.4 phosphate buffered saline (PBS) were explored, and the parabolic diffusion model simulate the release progress best, showing that the release process belongs to multi phase diffusion process via ion exchange. At last, the anticancer efficacy of all MTX/LDHs hybrids was also estimated by MTT assay with the human lung cancer (A549). It is found for the first time that the drug efficacy is closely associated with dispersion coefficient (noted as ϵ).

[Analysis of genotype IV distribution of hepatitis E virus infections in Wuhan by sequencing the open reading frame 3 gene].

OBJECTIVE: To determine the distribution of genotype IV among hepatitis E virus (HEV) infections in Wuhan by sequencing the open reading frame (ORF) 3 gene of HEV clinical isolates. METHODS: Serum samples were collected from 103 individuals who tested positive for the anti-HEV IgM antibody, and total HEV RNA was extracted for targeted gene sequencing analysis. Reverse transcription-nested polymerase chain reaction (PCR) was used to amplify two fragments of the ORF3 gene (5020 to 5392 nt and 5347 to 5956 nt, EF570133). The two PCR products were sequenced and the sequences were stitched with the ContigExpress program and used to determine the HEV genotype. RESULTS: Both ORF3 gene fragments were amplified in 18 out of the 103 anti-HEV IgM-positive serum samples. These 18 HEV isolates shared 92.5% to 99.4% identity with each other at the nucleotide level. Nucleotide sequence homology analysis of the HEV genotypes I, II, III, and IV indicated the highest homology was with genotype IV; specifically, homology with genotype I was 83.5% to 86.7%, with genotype II was 83.2% to 85.2%, with genotype III was 84.6% to 87.2%, and with genotype IV was 92.0% to 96.5%. CONCLUSION: Targeted sequencing of the HEV ORF3 gene facilitated genotyping of clinical isolates. Using this method, it was determined that nearly 20% of HEV clinical isolates from Wuhan belong to genotype IV.

Role of Hedgehog signaling pathway in proliferation and invasiveness of hepatocellular carcinoma cells.

The Hedgehog (Hh) pathway has been confirmed a contributor to the carcinogenesis and progression of various tumor types. To investigate the Hh signaling activity in human hepatocellular carcinoma (HCC), we detected the expression levels of Hh pathway components (Shh, Ptch1 and Gli2) in 57 samples of HCC and corresponding adjacent-tumor liver tissues. The Hh pathway was overexpressed in cancer tissues compared with non-cancer tissues and correlated closely with histologic differentiation and portal venous invasion of HCC. To elucidate the relationship between Hh signaling and HCC progression, we performed a further study in vitro. First, the expression levels of the signaling were detected in a subset of hepatoma cell lines and SMMC-7721 cells selected with high level of Hh signaling expression. Next, we employed KAAD-cyclopamine (a specific inhibitor of Hedgehog pathway) to block the Hh pathway in SMMC-7721 cells and assessed the changes of their biological behaviors. The results showed that the blockade of Hh signaling pathway by KAAD-cyclopamine induced reduction of DNA synthesis leading to marked cell growth inhibition and also caused significant attenuation in invasiveness and motility of HCC cells. Collectively, our data demonstrated that the Hh pathway plays an important role in HCC development and invasion. Blockade of the Hh signaling pathway may be a potential target of new therapeutic strategy for HCC.

[The effect of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure].

OBJECTIVES: To explore the effects of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure. METHOD: Twenty-four healthy male SD rats were randomly divided into four groups (6 rats in each group) and all of them were injected intraperitoneally with solutions: group I with normal saline, group II with 400 mg/kg of D-galactosamine (D-GaLN), group III with 400 mg/kg of D-GaLN plus 50 microg/kg lipopolysaccharide(LPS), and group IV with 400 mg/kg of D-GaLN plus 500 microg/kg LPS. At 6 hours after the administration of different solutions intraperitoneally, blood samples were collected to examine blood urea nitrogen (BUN) and serum creatinine. Realtime PCR was used to study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the livers and kidneys. RESULTS: No endotoxemia developed in group I or group II but it was evident in group III and group IV. The level of endotoxemia in group IV was higher than in group III (8.05+/-0.43, 3.50+/-2.25, P<0.05). After 6 hours of administration of LPS in group IV, hypoglycemia appeared, and blood glucose was normal in the other three groups. BUN and serum creatinine were all normal in the four groups, except that blood urea nitrogen was elevated in group IV. The mRNA of PEPCK in livers decreased gradually in all the four groups (2.54+/-1.32 vs 1.87+/-0.15 vs 0.91+/-0.13 vs 0.44+/-0.42, P<0.05). In the kidneys there was no change in the expression of PEPCK in group I and group II (0.75+/-0.03 and 0.77+/-0.04, P>0.05), but it increased in group III (0.75+/-0.03 vs 1.63+/-0.86, P<0.05), and decreased in group IV (0.75+/-0.03 vs 0.13+/-0.07, P<0.05). CONCLUSION: During acute hepatic failure severe endotoxemia would damage the function of gluconeogenesis in livers and kidneys by inhibiting transcription of PEPCK and this can induce hypoglycemia.

[Efficacy and safety of reduced osmolarity oral rehydration salts in treatment of dehydration in children with acute diarrhea--a multicenter, randomized, double blind clinical trial].

OBJECTIVE: To assess the efficacy and safety of reduced osmolarity oral rehydration salts (ROORS) in treatment of mild to moderate dehydration caused by acute diarrhea in children. METHODS: A multicenter, randomized, double-blind, positive drug controlled clinical trial was conducted in 125 cases aged 1 to 17 years. These children with acute diarrhea and signs of dehydration were randomly assigned to receive either ROORS (trial group, n = 62) or oral rehydration salts II (ORS II) (control group, n = 63). The volume of intravenous infusion were recorded. The improvements of systemic symtoms and signs, diarrhea, dehydration and total scores were compared between the two groups. The adverse events and changes of electrolyte and other laboratory tests during treatment were also observed and analyzed. RESULTS: The overall effective rates in trial group and control group were 96.8% and 96.8%, respectively. The recovery of systemic symptoms, dehydration signs and diarrhea occurred in 96%, 97% and 78% patients in trial groups, and 96%, 98% and 85% patients in control group. The scores of symptoms and signs in both groups decreased significantly after treatment. All the above parameters and the number of cases who needed intravenous infusion (41 vs. 39) were not statistically different between two groups. However, the average volume of intravenously infused fluids in trial group was (450.98 +/- 183.07) ml, 24.5% less than that in the control group (597.30 +/- 343.37) ml (P < 0.05). The mean serum Na(+) concentration elevated from (137.48 +/- 4.55) mmol/L to (139.52 +/- 3.25) mmol/L (P < 0.01) in control group after treatment, but the change was not statistically significant in trail group. Serum K(+), Cl(-), HCO(3)(-) and other laboratory result did not change significantly after treatment. The total scores in both groups decreased obviously after treatment, but no significant difference was demonstrated between two groups (P > 0.05). A case in trial group had mild abdominal distention and recovered spontaneously. CONCLUSION: ROORS was shown to be effective and safe in the treatment of mild and moderate dehydration induced by acute diarrhea. Compared to ORS II, ROORS could decrease the intravenous supplement of fluid and lower the risk of hypernatremia.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice.

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 x 10(6)) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 microg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-gamma and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% +/- 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 +/- 0.093 vs 1.081 +/- 0.028, P < 0.01), the level of IFN-gamma secreted by splenocytes was higher (266.575 +/- 51.323 vs 135.223 +/- 32.563, P < 0.01) , and IL-4 level was lower (22.385 +/- 2.252 vs 40.598 +/- 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Significance of serum IgA in patients with acute hepatitis E virus infection.

AIM: To study the significance of serum anti-hepatitis E virus (HEV) IgA in patients with hepatitis E. METHODS: A new method was established to assay anti-HEV IgA, which could be detected in the middle phase of the infection. We compared anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG assay in sera from 60 patients with positive HEV-RNA. RESULTS: The 60 patients with positive HEV-RNA had both anti-HEV IgA and anti-HEV IgM and 410 patients with negative HEV-RNA were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNA was detectable in the serum until 20 +/- 11 d. We used anti-HEV IgM and anti-HEV IgA assay to detect HEV infection and positive results were found in 90 +/- 15 d and 120 +/- 23 d respectively, the positive rate of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA (P < 0.05). CONCLUSION: The duration of anti-HEV IgA in serum is longer than that of anti-HEV IgM, and anti-HEV IgA assay is a good method to detect HEV infection.

[Infection of tupaia hepatocytes with hepatitis C virus in vitro].

OBJECTIVE: Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro. METHODS: Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage. RESULTS: Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage. CONCLUSION: The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.

Epidemiology and genotypes of HEV in Wuhan.

BACKGROUND: Understanding the genotype and clinical features of the hepatitis E virus (HEV) are important for understanding its characteristics, for evaluating region-specific diagnostic assays, and producing vaccines. OBJECTIVES: To investigate the epidemiology and the genotypes of HEV among outpatients and inpatients in the Department of Infectious Diseases of Tongji Hospital in Wuhan, China. METHODS: Clinical data were elicited from the hospital records of patients who were clinically diagnosed with acute hepatitis between January 2000 and August 2004 (4920 patients). Of these cases, 120 patients with anti-HEV-IgM, IgG-positive were selected to analysis. Conserved genomic sequences of open reading frame 2 (345 bp) in the HEV gene were detected using polymerase chain reaction, 25 of which were cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysis of genotypes strains. RESULTS: The HEV infection rate is gradually increasing in Wuhan. The number of male patients was 3.3-fold greater than the number of female patients found in clinical investigations. People aged 30-59 years are more susceptible to infection, and people are more susceptible in March-June. Twenty-five isolates shared the same genotype, genotype IV, with 82.61-98.55% nucleotide identity. This genotype had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology with the nucleotide sequence of HEV genotypes I-IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented at least three different subtypes, but there were no significant differences found in the epidemiological features or liver function of patients with the three subtypes. CONCLUSIONS: HEV sequences isolated from patients in Wuhan belong to different subtypes of HEV genotype IV.

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice.

AIM: To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide (TAA). METHODS: Twenty-four male C57Bl/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline (2 mL/kg), leptin (1 mg/kg), TAA (200 mg/kg), TAA (200 mg/kg) plus leptin (1 mg/kg) respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and superoxide dismutase (SOD), malondialdehyde (MDA) in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin (HE) staining and picric acid-Sirius red dyeing were performed. The level of alpha1(I) procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS: Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group (205.67+/-27.69 U/L vs 50.67+/-10.46 U/L, 177.50+/-23.65 U/L vs 76.33+/-12.27 U/L, 2.60+/-0.18 nmol/mg pro vs 1.91+/-0.14 nmol/mg pro, P<0.01) and in TAA plus leptin group (256.17+/-22.50 U/L vs 50.67+/-10.46 U/L, 234.17+/-27.37 U/L vs 76.33+/-12.27 U/L, 2.97+/-0.19 nmol/mg pro vs 1.91+/-0.14 nmol/mg pro, P<0.01). The level of SOD in livers decreased (51.80+/-8.36 U/mg pro vs 81.52+/-11.40 U/mg pro, 35.78+/-6.11 U/mg pro vs 81.52+/- 11.40 U/mg pro, P<0.01) and the level of alpha1(I) procollagen mRNA in liver tissues also increased (0.28+/-0.04 vs 0.11+/- 0.02, 0.54+/-0.07 vs 0.11+/-0.02, P<0.01). But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and alpha1(I) procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased (256.17+/-22.50 U/L vs 205.67+/- 27.69 U/L, P<0.05; 234.17+/-27.37 U/L vs 177.50+/-23.65 U/L, P<0.05; 2.97+/-0.19 nmol/mg pro vs 2.60+/-0.18 nmol/mg pro, P<0.05; 0.54+/-0.07 vs 0.28+/-0.04, P<0.01; 3.17 vs 2.00, P<0.05), and the level of SOD in liver decreased (35.78+/-6.11 U/mg pro vs 51.80+/-8.36 U/mg pro, P<0.05). There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION: Leptin can exacerbate the degree of TAA-induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). METHODS: The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.