ABSTRACT
Objective To construct a bicistronic expression vector containing HPV type 6b L1 gene, to express the recombinant vector in mammalian cells, and to establish a cell strain stably expressing HPV6b L1 gene. Methods After endonuclease digestion and purification, the gene fragment of HPV6b L1 was cloned into the eukaryotic expression vector pIRES2-enhanced green fluorescent protein (EGFP). The identification of the recombinant was realized via endonuclease digestion and sequence analysis. Then, the recombinant plasmid pIRES2-HPV6bLl-EGFP was transfected into NIH3T3 (a mouse embryonic fibroblast cell line) cells. Subsequently, the expression of EGFP was observed by fluorescent inverted microscopy, and HPV6b L1 mRNA expression by reverse transcription (RT)-PCR. Results The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed, transfected into N1H3T3 cells, and selected by G418. The expression of EGFP was seen under an inverted fluorescence microscoy. RT-PCR proved the expression of HPV6b LI mRNA in transfected cells. Conclusions The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed and transfected into NIH3T3 cells. Inverted fluorescent microscopy and RT-PCR confirmed the successful expression of HPV6b L1 in NIH3T3 cells.
ABSTRACT
Objective To investigate the effects of intense pulsed light (IPL) irradiation on the content of collagen fibers, elastic fibers and hyaluronic acid in Kunming mouse skin. Methods The dorsal skin of mice was divided into two areas: the right area was irradiated with IPL, and the left remaining unirradiated served as the control. Skin specimens were taken from the back of mice on day 1, 3, week 1, 2, 4 and 8 after the irradiation and subjected to staining with HE, sirius red and Gomori aldehyde-fuchsin for examinations of histological changes, type Ⅰ and Ⅲ collagen fibers and elastic fibers. The hydroxyproline and hyaluronic acid content in skin tissues of mice was determined with ultraviolet spectrophotometry and radioimmunoassay respectively. Results After irradiation, a significant increase was observed in dermal thickness on week 2 (t =4.623, P＜ 0.05), 4, and 8 (t = 3.904, P＜ 0.05), in type Ⅲ collagen fiber (t = 5.129, P＜ 0.05) on week 1,in type Ⅰ and Ⅲ collagen fibers on week 2, 4 and 8 (both P＜ 0.05), in elastic fibers from week 2 to 8 (P ＜0.05), and in hydroxyproline content from week 1 to 8 (all P ＜ 0.05) in the skin of mice compared with unirradiated mice. In detail, dermal thickness increased by 18.71% on week 4, and type Ⅲ collagen fiber by 40.54% in irradiated mice compared with unirradiated mice. Further more, the hyaluronic acid content was elevated from day 1 to 3, but gradually declined from week 1 to 8, and remained statistically higher from day 1 to week 8 (P ＜ 0.05) in irradiated mice compared to unirradiated mice. Conclusion IPL irradiation could induce an increase in the content of collagen fiber, elastic fiber and hyaluronic acid in the dorsal skin of mice.
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Objective To investigate the expressions of survivin, cyclooxyenase-2 (COX-2) and vascular endothelial growth factor (VEGF) and their relationship with angiogenesis in condyloma acuminatum (CA) tissues. Methods Immunohistochemistry using PowerVision staining kit was performed to detect the expression of survivin, COX-2 and VEGF protein in 60 CA tissue samples from patients and 21 normal skin samples from the foreskin of human controls. At the same time, the microvessel density was determined in CA tissues by staining blood vessel endothelium with anti-CD105 monoclonal antibody. Results The positivity rate of survivin and COX-2 expression was 56.67% and 63.33%, in CA tissues, 9.52% and 0 in normal skin tissues, respectively. There was a significant difference between the two groups of tissue samples in the positivity rate and intensity of survivin and COX-2 expression (all P < 0.05). VEGF was expressed in all of the CA tissues and normal skin tissues, while the intensity of VEGF expression was statistically different between the two groups of tissue samples (P < 0.05). The MVD was 16.38 ± 5.46 and 0.62 ± 0.44 in CA tissues and normal skin tissues, respectively (P < 0.05). There was a significant positive correlation between the expressions of survivin, COX-2 and VEGF, as well as between MVD and the expressions of survivin and COX-2 in CA tissues. Conclusion The expression levels of survivin, COX-2 and VEGF are significantly higher in CA tissues than in normal skin tissues.
ABSTRACT
Objective To assess the mutation in exon 8 of C1 esterase inhibitor(C1INH)gene in a patient with hereditary angioedema(HAE).Methods Genomic DNA was extracted from a female patient with HAE as well as her mother and a normal human control.The fragment of exon 8 of C1INH gene was amplified by PCR and inserted into plasmid carrier pUC19 with the help of ligase.Then,the recombinant plasmid was transformed into competent cells of E coli TG1 strains.After culture of positive transformant,plasmid DNA Was extracted and subjected to sequencing.SDS-PAGE and We:stem blot were performed on the sera of the patient to detect the concentration and function of C1INH protein.Results An A1677G mutation at exon 8 of C1INH gene.which resulted in a substitution of isoleucine to valine at codon 440,Was found in the patient who SUfiered from HAE type I.Additionally.SDS-PAGE and Western blot revealed that the molecular weight of C1INH protein was 96 000.but not 105 000 observed in noHnal human control.Conclusion The newly identified mutation 1440V.which is located at P4 residue of reactive center loop in C1INH.may result in conformational alteration of C1INH.