[Effects of miR-122 on expression of hepatitis B virus proteins].

OBJECTIVE: To investigate the effect of miR-122 on the expression of hepatitis B virus (HBV) proteins. METHODS: Anti-sense oligodeoxynucleotide (ASODN) of two different sequences against miR-122, anti-miR-122 and LNA-antimiR-122 (Locked nucleic acid), human miR -122 (hsa-miR-122), or the negative control anti-GFP were designed and synthesized, then transfected into HepG2.2.15 cells. After 24 h and 48 h, the levels of HBsAg and HBeAg in the supernatant were determined with a time-resolved immunofluorometric assay (TRFIA). HBV DNA in supernatant and miR-122 in cells were measured by quantitative real-time PCR. RESULTS: After 48 h expressions of miR-122 in the LNA-antimiR-122 and anti-miR-122 groups were significantly suppressed and lower than those in the negative control (P<0.001), while the level of miR-122 in the hsa-miR-122 group was higher than that in the negative control (P<0.001). The expression of HBeAg and HBsAg in hsa-miR-122 group was lower than that in the negative control (P<0.01) 24 h and 48 h after transfection. The expression of HBeAg and HBsAg in the anti-miR-122 group and LNA-antimiR-122 group was significantly lower than that in negative control (P>0.001). The levels of viral DNA at both time-points in the various test groups were not significantly different from those of negative control group (P>0.05). CONCLUSION: miR-122 may regulate HBV antigens and potentially affect the progress of pathogenesis, which might be the new targets for treatment of HBV infection.

[Inhibition of hepatitis B virus S gene and C gene expression by different 10-23 DNAzymes substrate-recognition domains].

OBJECTIVE: To explore the inhibition effects of 10-23 DNAzymes with different substrate-recognition domains targeting hepatitis B virus (HBV) S gene and C gene expression in 2.2.15 cells. METHODS: 10-23 DNAzymes with different substrate-recognition domains specific to HBV S gene open reading frame (ORF) A(157)UG and HBV C gene ORF A(1816)UG were designed and synthesized, respectively. Different 10-23 DNAzymes were transfected into 2.2.15 cells which is a stable HBV producing cell line. HBsAg and HBeAg secreted into culture media were detected by radioimmunoassay (RIA) and HBV DNA levels were measured by real-time PCR. 3-(4, 5-dimethylthiagol-2-yl)-2, 5-drphnyl tetrazolium bromide (MTT) assays were performed to evaluate cytotoxicity. RESULTS: HBsAg and HBeAg expressions were reduced by various DNAzymes (0.1 - 2.5 micromol/L) with different substrate-recognition domains after transfection. The antiviral effects of DNAzymes were apparent until 72 h post-transfection. The inhibition rates of the DNAzymes at the same dose on HBsAg and HBeAg in the same period of post-transfection were as the following: DrzBS-9 > DrzBS-8 > DrzBS-7; DrzBC-9 > DrzBC-8 > DrzBC-7. Among all the DNAzymes used, DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent, with HBsAg and HBeAg reduced 95% and 92% 48 h post-transfection at the dose of 2.5 micromol/L, respectively. The inhibition effects on HBV DNA by various DNAzymes with different substrate-recognition domains were of no significance. There were no evident cytotoxic effects of these DNAzymes in the range from 0.1 to 2.5 micromol/L. CONCLUSION: 10-23 DNAzymes with different substrate-recognition domains targeting HBV S gene and C gene mRNA possessed specific inhibition effects in 2.2.15 cells, and DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent.

Construction and application of a yeast expression system for thymosin a1

We want to construct a yeast expression system for thymosin a1 (Ta1) to make the orally administered Ta1 preparation possible. The whole Ta1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Ta1 in recombinant coincided with the original one reported in Genbank. When pYES2-Ta1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Ta1 expression. Western blot was performed to identify the quality of the expressed Ta1. Dried yeast containing pYEST2-Ta1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Ta1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Ta1 and synthesized Ta1 peptide can significantly increase the CD8+ level (22.74±1.09 and 18.77±4.72 vs 7.49±2.14, p<0.01), while both of them had little effect on the CD4+ lymphocytes (61.86±6.94 and 65.91±4.78 vs 57.93±10.40, p>0.05). We concluded that a high effective yeast expression system for Ta1 was constructed successfully and the Ta1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

Immunomodulatory function of orally administered thymosin alpha1.

OBJECTIVE: To investigate the immunological function of a yeast expression system for thymosin alpha1 (Talpha1). METHODS: A constructed Talpha1 yeast expression system was used to investigate the immunological function of orally administered Talpha1. Dried yeast containing three different concentration of Talpha1 was fed to normal Balb/c mice and other Balb/c mice whose immunities were inhibited in advance by cyclophosphamide. Synthesized Talpha1 peptide was used as positive control and dried yeast with empty plasmid was used as negative control. CD4(+) and CD8(+) levels were detected by flow cytometry assay. TNF-alpha, IFN-gamma, IL-2, IL-6 and IL-10 levels were detected by liquid chip. RESULTS: In normal Balb/c mice or immune inhibition Balb/c mice, CD8(+) levels were significantly increased. Especially in immune inhibition Balb/c mice, CD8(+) levels in synthesized Talpha1 group (18.77%+/-4.72%), small dose group (13.48%+/-6.17%) and large dose group (22.74%+/-1.09%) were significantly higher than that in empty yeast control group (7.49%+/-2.14%). CONCLUSION: Orally administered Talpha1 has its certain immunomodulatory function.

Construction and application of a yeast expression system for thymosin alpha1.

We want to construct a yeast expression system for thymosin alpha1 (Talpha1) to make the orally administered Talpha1 preparation possible. The whole Talpha1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Talpha1 in recombinant coincided with the original one reported in Genbank. When pYES2-Talpha1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Talpha1 expression. Western blot was performed to identify the quality of the expressed Talpha1. Dried yeast containing pYEST2-Talpha1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Talpha1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Talpha1 and synthesized Talpha1 peptide can significantly increase the CD8+ level (22.74 +/- 1.09 and 18.77 +/- 4.72 vs 7.49 +/- 2.14, p < 0.01), while both of them had little effect on the CD4+ lymphocytes (61.86 +/- 6.94 and 65.91 +/- 4.78 vs 57.93 +/- 10.40,p > 0.05). We concluded that a high effective yeast expression system for Talpha1 was constructed successfully and the Talpha1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

Construction and application of a yeast expression system for thymosin alpha1.

We want to construct a yeast expression system for thymosin alpha1 (Talpha1) to make the orally administered Talpha1 preparation possible. The whole Talpha1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Talpha1 in recombinant coincided with the original one reported in Genbank. When pYES2-Talpha1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Talpha1 expression. Western blot was performed to identify the quality of the expressed Talpha1. Dried yeast containing pYEST2-Talpha1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Talpha1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Talpha1 and synthesized Talpha1 peptide can significantly increase the CD8+ level (22.74 +/- 1.09 and 18.77 +/- 4.72 vs 7.49 +/- 2.14, p < 0.01), while both of them had little effect on the CD4+ lymphocytes (61.86 +/- 6.94 and 65.91 +/- 4.78 vs 57.93 +/- 10.40,p > 0.05). We concluded that a high effective yeast expression system for Talpha1 was constructed successfully and the Talpha1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

[Serum cytokine profiles in stable survivors with clinical liver transplantation].

OBJECTIVE: To elucidate the profile of serum cytokines and adhesion molecules in stable survivors with clinical liver transplantation. METHODS: Flow cytometric analysis was used to analyse the phenotype of T cell subsets in peripheral blood mononuclear cells (PBMCs) from group of liver transplantation (LTx) (n = 22), primary liver carcinoma (PLC) (n = 13) and healthy control (n = 12). Enzyme-linked immunoabsorbent assay (ELISA) was used to determine the serum cytokines and adhesion molecules profiles in stable survivors with clinical liver transplantation. RESULTS: Percentage of CD3(+) T cell and CD8(+) T cell, as well as ratio of CD4(+) to CD8(+) revealed no difference among three groups. The percentage of CD3(+)CD25(+) T cells in LTx group was found higher than that in healthy group (P = 0.022). Th1 cytokines (IL-2, IFN-gamma) and Th2 cytokines (IL-4, IL-10), as well as TNF-alpha displayed no significant difference among three groups. The levels of IL-6, ICAM-1 and P-selectin in serum were not found any difference between LTx group and PLC group, while the levels of IL-6, ICAM-1 and P-selectin in serum shown significant difference between LTx and healthy groups (P = 0.048, 0.000 and 0.025, respectively). CONCLUSIONS: Our data demonstrates that effector T-cells can also be activated and exert immunoresponse to grafts permanently under the treatment of immunosuppressant. Adhesion molecules (ICAM-1, P-Selectin) and pro-inflammatory cytokines (IL-6, TNF-alpha) might be involved in the process of chronic graft damage induced by allo-immunoresponse.

[The diagnostic value of eight serum indices for liver fibrosis].

OBJECTIVES: Using receiver operating characteristic (ROC) curve to compare the value of platelet derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (MMP-1), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hyaluronic acid (HA), type III procollagen (PCIII), type IVcollagen (C-IV) and laminin (LN) in serum and message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononucleocytes (PBMC) to diagnose liver fibrosis. METHODS: Serum samples from 60 chronic hepatitis B patients and 20 healthy blood donors were assayed for serum level of PDGF-BB, TGF-beta1, MMP-1 and TIMP-1 with ELISA, and for serum level of HA, PCIII, C-IV and LN, with RIA. The mRNA expression of TIMP-1 and MMP-1 in PBMC was detected by RT-PCR. Liver biopsy was performed in all those patients. The biopsy materials were examined histopathologically. RESULTS: Through the analysis by ROC curve, serum PDGF-BB is the most valuable marker, and its sensitivity was the highest among eight indices. The marker with the highest specificity was TIMP-1 mRNA in PBMCs. The area under the curve (AUC) of PDGF-BB, TIMP-1, HA, PCIII, C-IV, LN, TIMP-1 mRNA was 0.985, 0.726, 0.318, 0.728, 0.727, 0.583, 0.463, 0.876, respectively. The sensitivity and specificity of combination of PDGF-BB and TIMP-1 mRNA were 97.4%, 95.0%, respectively. CONCLUSION: Serum PDGF-BB is the most potential index among eight markers. The combination of serum PDGF-BB and TIMP-1 mRNA in PBMC might be more efficient to screen liver fibrosis.

Diagnostic value of platelet derived growth factor-BB, transforming growth factor-beta1, matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis.

AIM: Noninvasive diagnosis of hepatic fibrosis has become the focus because of the limited biopsy, especially in the surveillance of treatment and in screening hepatic fibrosis. Recently, regulatory elements involved in liver fibrosis, such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been studied extensively. To determine whether these factors or enzymes could be used as the indices for the diagnosis of hepatic fibrosis, we investigated them by means of receiver operating characteristic (ROC) curve. METHODS: Serum samples from sixty patients with chronic viral hepatitis B and twenty healthy blood donors were assayed to determine the level of PDGF-BB, TGF-beta1, MMP-1, and TIMP-1 with ELISA, and HA, PCIII, C-IV, and LN level with RIA. The message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR and Northern blot hybridization. Liver biopsy was performed in all patients. The biopsy samples were histopathologically examined. The trial was double-blind controlled. RESULTS: The serum level of PDGF-BB, TIMP-1, the ratio of TIMP-1 and MMP-1 (TIMP-1/MMP-1), mRNA expression of TIMP-1 (TIMP-1mRNA), and the ratio of TIMP-1mRNA and MMP-1mRNA (TIMP-1mRNA/MMP-1mRNA) in patients was significantly higher than those in the healthy blood donors (t=2.514-11.435, P=0.000-0.016). The serum level of PDGF-BB, TIMP-1, TIMP-1/MMP-1, and TIMP-1mRNA was positively correlated with fibrosis stage and inflammation grade (r=0.239-0.565, P=0.000-0.033), while the serum level of MMP-1 was negatively correlated with fibrosis stage and inflammation grade, and TIMP-1mRNA/MMP-1mRNA was positively correlated with inflammation grade. Through the analysis by ROC curve, serum PDGF-BB was the most valuable marker, and its sensitivity was the highest among the nine indices. The markers with the highest specificity were TIMP-1mRNA and TIMP-1mRNA/MMP-1mRNA in PBMCs. The area under the curve (AUC) of PDGF-BB, TIMP-1mRNA, TIMP-1mRNA/MMP-1mRNA, TIMP-1/MMP-1, HA, PCIII, TIMP-1, C-IV, and LN was 0.985, 0.876, 0.792, 0.748, 0.728, 0.727, 0.726, 0.583, and 0.463, respectively. The sensitivity and the specificity in the parallel test was 99.0% and 95.0% when serum PDGF-BB, TIMP-1mRNA and TIMP-1mRNA/MMP-1mRNA was detected simultaneously. CONCLUSION: Serum level of PDGF-BB, TIMP-1mRNA, TIMP-1mRNA/MMP-1mRNA in PBMCs, and serum level of TIMP-1 and TIMP-1/MMP-1 can be used as the indices for the diagnosis of hepatic fibrosis, but the former three are more useful. The combination of serum PDGF-BB, TIMP-1mRNA and TIMP-1mRNA/MMP-1mRNA in PBMCs is even more efficient in screening liver fibrosis.

[Value of serum-vascular endothelial growth factor in the differential diagnosis of solitary pulmonary nodule].

OBJECTIVE: To evaluate serum-vascular endothelial growth factor (S-VEGF) in the differentiation of solitary pulmonary nodule (SPN). METHODS: Serum level of VEGF of 68 patients with SPN was measured by ELISA kit, and compared with the control group of 20 normal subjects. The nodules were diagnosed by operation and pathology. RESULTS: The median level of S-VEGF was 42.5 (range from 10 to 170) pg/ml in the control, 44 (range from 18 to 360) pg/ml in benign nodule group and 75 (range from 18 to 890) pg/ml in lung cancer group, with significant difference observed between the nodule group and control (P < 0.01), and between the lung cancer group and the benign nodule group (P < 0.05), but not between the benign nodule group and the control. In addition, when S-VEGF in different pathologic types of the limited number of lung cancer patients were compared, no significant difference was observed. CONCLUSION: S-VEGF is valuable in the differential diagnosis of solitary pulmonary nodule. An elevated S-VEGF level >or= 100 pg/ml in patients with SPN may strongly speak for a malignant nodule. Operation is suggested.

[Modulation and implications in pathogenesis of interferon gamma receptor 1 in patients with chronic hepatitis B].

OBJECTIVE: To study the possible differences in the interferon gamma receptor (IFN-gamma R1) response among a variety of clinical types in patients with chronic hepatitis B (CHB) and implications in pathogenesis. METHODS: The serum level of IFN-gamma and the expression level of IFN-gamma R1 in peripheral leucocytes, from 53 CHB patients, were examined by ELISA and flow cytometry respectively, which were compared with the baseline levels of 15 healthy controls and were performed correlation analysis with alanine aminotransferase (ALT), total bilirubin (TBil) in serum and morphological change in hepatic tissues. RESULTS: The results showed that the level of IFN-gamma R1 (28.89% 11.77%) expressed on the membranes of lymphocytes in CHB patients, which correlated with liver inflammation (r=0.621, P<0.01) and serum TBil level (r=0.575, P<0.01), was much higher than that (9.23% 1.30%) of the healthy controls (Z=3.988, P<0.05), and no obvious difference on the membranes of leucocytes. The serum levels of IFN-gamma in patients with cirrhosis and severe hepatitis were higher than those of healthy controls. And the two was no difference from each other, but the standard deviation in each group was relatively large. CONCLUSION: These findings suggest that the IFN-gamma signal transduction pathway is modulated through up-regulating the expression of IFN-gamma R1 on the membranes of lymphocytes, which takes part in the immuno-pathogenesis in CHB patients.