Transfecting keratinocyte and enhancing cell proliferation by integrated HPV11 DNA / 生物工程学报

We constructed the plasmid containing the integrated open reading frame (ORF) HPV11 genome, and identified its function, to lay foundation for production of transgenic animal models of HPV11. Recombinant plasmid pQE-Trisystem- EGFP/HPV11 (pE/H) and pQE-Trisystem-EGFP/1.1 copy HPV11 (pE/1.1H) were constructed. Then, the human keratinocyte (KC) was transfected by pE/1.1H, pE/H and closed circular HPV11 and detected. The recombinant plasmid was successfully constructed. The expression of HPV11E6 gene was detected in the experimental group. Fluorescence was observed in the pE/1.1H and pE/H group. The HPV11, pE/H, and pE/1.1 enhanced the KC proliferation, with remarkable differences (P < 0.01) from the control group. Amongst the three experimental groups, pE/1.1H was found to be the weakest. The plasmid containing the integrated ORF of HPV11 (1.1 copy HPV11) genome was successfully constructed. The pE/1.1H had the same phenotype of wild-type HPV11 genome. It provided experimental material and methodological guidance for studying the low-risk HPV, as well as for the production of HPV11 transgenic mice.

Calpain-mediated Cleavage of Atg5 Determine Autophagy or Apoptosis of PMN, and C5a's Role / 生物化学与生物物理进展

Apoptosis of neutrophils controls the duration and the intensity of an inflammatory response and therefore the extent of neutrophil- mediated tissue damage, disturbance of neutrophil apoptosis has been associated with many diseases, underlying mechanism is not elucidated. C5a is a complement fragment that has multifunctional properties, which induces neutrophil chemoattraction, an oxidative burst, enhancement of phagocytosis, release of granule enzymes, and suppress neutrophil apoptosis. Several studies have reported calpain is involved in both neutrophil functions and apoptosis and it might play a more specific role in the regulation of neutrophil apoptosis. Diffenrent isoform of calpains is activted by diffenrent stimuli through different transduction pathway. It was reported previously that calpain is required for neutrophil migration and chemotaxis induced by C5a. In addition, autophagy is a ubiquitous physiological process that occurs in all eukaryotic cells and is considered to be a survival mechanism. Atg5 promotes autophagy and is indispensable to autophagosome formation. Upon calpain activation, Atg5 is cleaved and the resulting 24 ku Atg5 mediates apoptosis while losting the property of autophagy. Therefore, Atg5 represents a molecular switch between autophagy and apoptosis. The interaction among the C5a, calpain and Atg5 was introduced and new direction for further research was provided.

The role of C5a in adhesion properties of polymorphonuclear leukocyte to pulmonary vascular endothelial cells in burn patients with acute lung injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the postburn adhesion properties of polymorphonuclear leukocyte (PMN) onto pulmonary vascular endothelial cells (PVEC) in burn patients with acute lung injury (ALI), so as to determine the role of C5a on PVEC-PMN adhesion.</p><p><b>METHODS</b>Microtubule sucking technique was employed to determine the PVEC-PMN adhesion. The myeloperoxidase (MPO) was also assayed to reflect the magnitude of PVEC-PMN adhesion.</p><p><b>RESULTS</b>The magnitude of PVEC-PMN adhesion increased and the adhesion force increased along with an increase in rh-C5a concentration. Simultaneously, the MPO activity was increased, which could be inhibited by anti-C5aR McAb in a concentration 1:104.</p><p><b>CONCLUSION</b>Both C5a and C5aR participated in PVEC-PMN adhesion, which might be important in the pathogenesis of ALI.</p>

Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells / 第三军医大学学报

Objective To construct a recombinant plasmid for expressing the HPV18 E2 gene and develop some materials for HPV18 related disease.Methods The E2 gene of HPV18 was amplified by PCR from PBR322-HPV18 and cloned into PIRES2-EGFP.The sequence of cloned HPV18E2 was confirmed by restriction analysis and DNA sequencing,then transfected into HeLa cells.E2 gene was detected by RT-PCR.Results HPV18 E2 gene fragment was amplified by PCR and ligated to PIRES2-EGFP,then transfected into HeLa cells successfully.The expression of green fluorescence cells was observed by fluorescence microscopy.Specific band could be seen in transfected cells by RT-PCR.Conclusion Recombinant plasmid PIRES2-EGFP is effectively expressed after being transfected into HeLa cells in vitro.

Inhibitive effect of peritoneal decoy-oligoligomucleotidesnucleotides targeting nuclear factor-?B on expression of inflammatory mediators in macrophages ? cells / 中华创伤杂志

Objective To design and synthesize targeting nuclear factor-?B (NF-?B) decoy-oligodeoxynucleotides (ODNs) and measure their anti-inflammatory actions. Methods Peritoneal macrophages ? (pM?) cells extracted from rats were randomly divided into normal control group, lipopolysaccharides (LPS)-stimulated group (Group A), decoy-ODNs treated group (Group B), non-decoy-ODNstreated group (Group C) and cationic liposome treated group (Group D). Supernatants and cells in the control group and the Group A were collected 1,2,6,12,18 and 24 hours after LPS stimulation. By using cationic liposomes in a ratio of 4:1 in quantity, pM? cells were transfected by decoy-ODNs at concentrations of 2 mg/L,4 mg/L and 8 mg/L respectively and stimulated with LPS 6 hours later. Then, supernatants and cells were collected 8, 12 and 18 hours after transfection. The expression changes and the distribution of p65 were analyzed by immunocytohistochemical method, the protein expressions of tumor necrosis factor ? (TNF?), interleukin-6 (IL-6) and interleukin-10 (IL-10) in the supernatants by enzyme-linked immunosorbent assay (ELISA) and mRNA transcriptions of TNF?,IL-6 and IL-10 by reverse transcriptase polymerase chain reaction (RT-PCR). Results Decoy-ODNs at concentration of 4 mg/L and 8 mg/L could markedly inhibit the expression and transcription of TNF? and IL-6 of pM? cells in a dose-dependent fashion but had a weak inhibitive effect on the expression and transcription of IL-10. Conclusions The targeting NF-?B Decoy-ODNs can suppress the expressions of inflammatory mediators in pM? cells.