Min-wei Li

Zhejiang University, Hang-hsien, Zhejiang Sheng, China

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Publications (16)18.79 Total impact

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    ABSTRACT: 10–23 DNAzyme (DrzBC) could block the expression of HBV e-gene with application limit of dependence on exogenous delivery. Stearic acid grafted chitosan oligosaccharide (CSSA) could self-aggregate to form micelles in aqueous medium and bind with DrzBC by electrostatic interaction. Compared with Lipofectamine™ 2000, the CSSA micelles showed much lower cytotoxicity (412.5μg/mL) and advantaged target in subcellular organelles-cytoplasm. Both including DrzBC of 1μmol/L, Lipofectamine™ 2000/DrzBC complex showed maximum inhibition rate (IR) on HBeAg expression of 46.53±2.00% at 48h, and then decreased rapidly, while CSSA/DrzBC complex showed maximum IR of 82.51±1.28% at 72h, and hold on IR above 70% until 96h. Moreover, within a concentration range of 0.1–2.0μmol/L, and equally incubating for 48h, the IR of Lipofectamine™ 2000/DrzBC complex was from 23.20±1.61% to 66.27±1.96%, while the IR of CSSA/DrzBC complex increased from 40.23±3.28% to 80.95±1.69%. CSSA/DrzBC complex is a promising effective system for inhibiting HBeAg expression.
    Carbohydrate Polymers 01/2012; · 3.92 Impact Factor
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    ABSTRACT: Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.
    Journal of virological methods 08/2009; 163(1):31-9. · 2.13 Impact Factor
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    ABSTRACT: Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.
    Vaccine 06/2009; 27(52):7451-8. · 3.77 Impact Factor
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    ABSTRACT: The influence of hepatitis B virus (HBV) gene heterogeneity on the failure of HBV vaccination in eastern China remains unknown. Here, we assigned 78 hepatitis B surface antigen (HBsAg)-carrier mothers to two groups: 41 mothers from whom transmission of HBV to their children was successfully prevented and 37 mothers whose children were HBsAg positive 1 year after HBV vaccination. The DNA loads in mothers of the failure group (4.17E + 07 copies/ml) were significantly higher than those in the success group (8.40E + 06 copies/ml). However, no difference was found in the S gene mutation rate and genotypes between the groups. Interestingly, Thr123Ala and Gly145Arg were observed only in failure-group mothers, whereas Thr126Asn, Thr126Ser, Thr143Asn, Asp144Gly, and Asp144Ala were seen in the success group. Thus, high viral load is an important risk factor for HBV vaccination failure, which is correlated with the positions of mutations in the S gene, but not with mutant frequencies or genotypes.
    Archives of Virology 03/2009; 154(3):437-43. · 2.28 Impact Factor
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    ABSTRACT: Herein, solid lipid nanoparticles (SLN) were proposed as a new drug delivery system for adefovir dipivoxil (ADV). The octadecylamine-fluorescein isothiocynate (ODA-FITC) was synthesized and used as a fluorescence maker to be incorporated into SLN to investigate the time-dependent cellular uptake of SLN by HepG2.2.15. The SLN of monostearin with ODA-FITC or ADV were prepared by solvent diffusion method in an aqueous system. About 15 wt% drug entrapment efficiency (EE) and 3 wt% drug loading (DL) could be reached in SLN loading ADV. Comparing with free ADV, the inhibitory effects of ADV loaded in SLN on hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) DNA levels in vitro were significantly enhanced.
    Journal of Zhejiang University SCIENCE B 07/2008; 9(6):506-10. · 1.11 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Zhonghua nei ke za zhi [Chinese journal of internal medicine] 05/2006; 45(5):396-9.
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    ABSTRACT: 10-23 DNA enzyme is one kind of deoxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2.2.15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A(1816)UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km, Kcat and Kcat/Km for Drz-HBV-C-9, were 1.4 X 10(-9) mol, 1.6 min-1 and 1.1 X 10(9) mol(-1) x min(-1), respectively. 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.
    Hepatobiliary & pancreatic diseases international: HBPD INT 12/2005; 4(4):573-6. · 1.26 Impact Factor
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    ABSTRACT: Interferon(IFN) with antiviral and immunomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNalpha-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells(CTLs) in patients with hepatitis B. Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNalpha-2a treatment. Before IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNalpha-2a treatment, showing no significant difference despite alternation of different degree. Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNalpha-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.
    Hepatobiliary & pancreatic diseases international: HBPD INT 09/2005; 4(3):403-5. · 1.26 Impact Factor
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    ABSTRACT: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.
    Journal of Zhejiang University SCIENCE B 08/2005; 6(7):664-7. · 1.11 Impact Factor
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    ABSTRACT: To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors. Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR. Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%. The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells
    Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 10/2004; 12(9):515-8.
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    ABSTRACT: To develop a system for quick screening of efficient siRNA targeted HBx mRNA. Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis. (1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388. Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.
    Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 08/2004; 33(4):300-5, 310.
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    ABSTRACT: To describe a novel mechanism for TRAIL up-regulation of CD4+, CD8+ T cells to participate in the pathophysiological process in patients with chronic hepatitis B (CHB). The serum levels of soluble TRAIL (sTRAIL), IFN-gamma and membrane bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 15 healthy controls, and correlation analysis were performed between ALT, TBil and PT, morphological change in hepatic tissues. The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than the healthy controls (P < 0.001), which of CD4+ T cells positively correlated with serum TBil (r=0.354, P = 0.008), Serum IFN-gamma level (r=0.302, P = 0.011) and which of CD8+ T cells positively correlated with serum TBil (r=0.522, P = 0.000), ALT (r=0.393, P = 0.003), PT (r=0.385, P = 0.004), serum IFN-gamma level (r=0.307, P = 0.009). The serum levels of soluble TRAIL only correlated with serum HBeAg expression (r=0.695, P = 0.001). These findings suggest that the expression of TRAIL on the membranes of lymphocytes was up-regulated, which may take part in the immunopathogenesis in CHB patients. TRAIL expression can be induced either by virus-specific protein expression or by inflammation cytokine IFN-gamma
    Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 05/2004; 12(5):284-6.
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    ABSTRACT: To investigate the effect of fragile histidine triad (FHIT) gene on the apoptosis of gastric cancer cells. FHIT gene was transfected into gastric cancer cell line MGC-803 by liposome. Western blot and immunohistochemical assay were employed to detect the expression of FHIT. Apoptosis and cell cycle were analyzed by flow cytometry (FCM). Morphological changes were observed by light and electron microscope. Stable expression of FHIT protein in transfected MGC-803 cells was obtained. The rates of G0/G1 phase and apoptosis cells were significantly higher in FHIT-transfected MGC-803 cells than those in control cells (64.4%compared with 49.5%and 32.0%compared with 12.2%, respectively). The results indicate that FHIT gene might be involved in the apoptosis of gastric cancer cells.
    Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 04/2004; 33(2):133-7.
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    ABSTRACT: It has been known that intra-cellular immunity is important for defense against viral infections and this function lies with interferon gamma (INF-gamma). Here we evaluated the role of IFN-gamma system in the pathogenesis of chronic hepatitis C (CHC). The levels of interferon gamma receptor alpha (IFNGR alpha) on the peripheral lymphocyte membrane were assayed with flow cytometry. The plasma concentrations of the cytokines IFN-gamma and IL-10 in CHC patients and normal controls were assayed by enzyme-linked-immunosorbent assay (ELISA). The samples were collected randomly from Xinjiang Autonomous Region, Zhejiang and the northern regions of Jiangsu Province in China. The levels of IFNGR alpha in CHC patients were significantly lower than that of normal controls (NC), especially among patients during the stable stage (P < 0.001), whereas there were no significant differences between CHC in active and stable stages. Among the patients of the three regions, there were no significant differences between patients from Xinjiang and Zhejiang provinces, but both had statistically significant difference compared with the patients from Jiangsu Province (P < 0.001). Plasma IFN-gamma and IL-10 concentrations in CHC patients decreased significantly, IFN-gamma in particular, but there were no significant differences in these levels between various stages of the disease. The IFN-gamma/IL-10 (Th1/Th2) ratio in patients was reversed. There may be defects in the IFN-gamma system in chronic HCV infected subjects and a low immune response, which may play an important role in the persistence of HCV infection.
    Chinese medical journal 01/2004; 117(1):79-82. · 0.90 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 01/2003; 11(1):14-6.
  • Chinese Journal of Integrative Medicine 02/2001; 7(1):29-29. · 1.06 Impact Factor