Construction and immunogenicity evaluation of chimerical DNA vaccine of human papillomavirus type 11 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct chimerical DNA vaccine plasmid of human papillomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity.</p><p><b>METHODS</b>Molecular cloning techniques were used to construct recombinant plasmid pcDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection.IL-2 and gamma-INF secreted by immunized spleens lymphocyte and HPV 11 L1 or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay.</p><p><b>RESULTS</b>The chimerical DNA plasmid of pcDNA3 L1-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and gamma-INF were detected in vaccinated mice.</p><p><b>CONCLUSION</b>Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.</p>

Effects of antisense epidermal growth factor receptor oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro.</p><p><b>METHODS</b>c-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method.</p><p><b>RESULTS</b>Compared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01).</p><p><b>CONCLUSION</b>The ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.</p>

Green tea polyphenol epigallocatechin-3-gallate inhibits the expression of nitric oxide synthase and generation of nitric oxide induced by ultraviolet B in HaCaT cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nitic oxide (NO) has been implicated in the pathogenesis of various inflammatory diseases, including sunburn and pigmentation induced by ultraviolet irradiation. Epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can protect skin from ultraviolet-induced damage. The purpose of this study was to investigate the protective mechanisms of EGCG on inducible nitric oxide synthase (iNOS) expression and NO generation by ultraviolet B (UVB) irradiation in HaCaT cells.</p><p><b>METHODS</b>HaCaT cells were irradiated with UVB 30 mJ/cm 2 and pretreated with EGCG at varying concentrations. The iNOS mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and NO production was quantified by spectrophotometric method. The expression of NF-kappaB P65 was measured by immunofluorescence cytochemistry staining.</p><p><b>RESULTS</b>The expression of iNOS mRNA and generation of NO in HaCaT cells were increased by UVB irradiation. EGCG down regulated the UVB-induced iNOS mRNA synthesis and NO generation in a dose dependent manner. The UVB-induced ctivation and translocation of NF-kappaB were also down regulated by EGCG treatment in HaCaT cells (P < 0.01).</p><p><b>CONCLUSIONS</b>Green tea derived-EGCG can inhibit and down regulate the UVB-induced activation and translocation of NF-kappaB, expression of iNOS mRNA and generation of NO respectively, indicating EGCG may play a protective role from UVB-induced skin damage.</p>

UVB-irradiated human keratinocytes and interleukin-1alpha indirectly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradiated dermal fibroblasts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.</p><p><b>METHODS</b>Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts.</p><p><b>CONCLUSIONS</b>UVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.</p>

Physical and chemical characters of recombinant human nucleoside diphosphate kinase A / 生物工程学报

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.

Effects of (-)-epigallocatechin-3-gallate on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibroblasts irradiated with ultraviolet A / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways. (-)-epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet-induced damage. In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro.</p><p><b>METHODS</b>Transcription factor Jun protein levels were measured by Western blot. Matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA were studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in conjunction with computer-assisted image analysis. MMP-1 and TIMP-1 proteins were quantified by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>EGCG decreased transcription activity of Jun protein after induction by UVA. Both the mRNA and protein levels of MMP-1 were increased by UVA irradiation, while no significant changes were observed in TIMP-1 levels. The ratio of MMP-1 to TIMP-1 showed statistically significant differences compared with the control. EGCG decreased the ratio of MMP-1 to TIMP-1 by inhibiting UVA-induced MMP-1 expression (P < 0.05).</p><p><b>CONCLUSION</b>EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP-1. The ratio of MMP-1 to TIMP-1, rather than the levels of MMP-1 or TIMP-1 alone, may play a significant role in human skin photodamage.</p>

Construction and Expression of Plasmid Coexpressing Human Papillomavirus Type 11 E7 and Human IFN?-2b / 中华皮肤科杂志

Objective To construct an expression plasmid of human papillomavirus type 11 E7 (HPV11-E7)/hurnan IFN?-2b fusion gene, to express the fusion gene in E.coli BL21, and pave way for further immunological study. Methods The recombinant plasmid was introduced into E.coli BL21, then the expression product was analyzed by SDS-PAGE and Western blotting after induction with isopropy-?-D-thiogalactoside (IPTG). Results The fusion gene of HPV11-E7 and human IFN?-2b was successfully cloned into pET-32a by a linker with the same sequence as we expected. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting. Conclusions The successful construction of prokaryotic expression plasmid and expression of HPV11-E7/human IFN?-2b fusion gene enable further immunological study.