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A 22-year-old female patient presented with skin flushing in the bilateral legs for 4 years, which gradually spread throughout the whole lower limbs and forearms 6 months ago. Skin examination showed diffuse flushing and dilated capillaries in the lower limbs and both forearms, and the flushing faded after a press. Histopathological examination of the skin lesion on the leg showed hyperkeratosis in a basket-like shape, increased pigmentation in the basal layer, infiltration of the superficial dermis with scattered lymphocytes, with no obvious red blood cell overflow; periodic acid-Schiff staining showed thickened and homogeneous deposits around the blood vessels; immunohistochemical staining showed thickened blood vessel walls and positive staining for type Ⅳ collagen. Diagnosis: cutaneous collagenous vasculopathy.
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Objective To investigate thymidylate synthase on pemetrexed treatment of lung adenocarcinoma effect relationship.Methods The 60 patients with lung adenocarcinoma in our hospital from January 2014 to January 2016 were selected as the research subjects.They were treated with pemetrexed.According to the clinical efficacy,they were divided into the effective group (n =27) and ineffective group (n =33) after 3 courses of treatment.The levels of thymidylate synthase (thymidylate synthase,TS),TS mRNA expression,and the expression of TS protein in the tumor tissues of two groups were analyzed by enzyme-linked immunoadsorbent assay (ELISA),fluorescence quantitative polymerase chain reaction (PCR),and immunohistochemistry.The relationship between TS level and pemetrexed in the treatment of lung adenocarcinoma was investigated.Results The level of ST in peripheral blood of the effective group was significantly lower than ineffective group.The objective response rate and protein of ST gene low expression were significantly higher than high expression of ST.Conclusions The level of thymidylate synthase in patients with adenocarcinoma of the lung is related to the therapeutic effect of pemetrexed in the treatment of adenocarcinoma of the lung.It can be used as a molecular marker to evaluate the clinical efficacy of pemetrexed in the treatment of patients with lung adenocarcinoma.
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Objective To evaluate the effect of selenomethionine (Se-Met) against ultraviolet B (UVB)-induced oxidative damage to human HaCaT keratinocytes,and to explore its possible mechanisms.Methods Cultured HaCaT cells were divided into several groups:normal control group receiving no treatment,Se-Met groups treated with Se-Met at concentrations of 1,10,50,100,200 nmol/L and 1 μmol/L for 24 hours respectively,UVB groups irradiated with UVB of 30,60 and 90 mJ/cm2 respectively,Se-Met + UVB groups treated with Se-Met at concentrations of 1,10,50,100,200 nmol/L and 1 μmol/L for 24 hours firstly,then irradiated with UVB of 30,60 and 90 mJ/cm2 respectively.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to estimate cellular proliferative activity,flow cytometry to detect cell apoptosis,colorimetry to evaluate superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and to determine malondialdehyde (MDA) levels.Statistical analysis was carried out by using factorial design analysis of variance (ANOVA),one-way ANOVA and least significant difference (LSD) test.Results Factorial design ANOVA showed that UVB radiation had an inhibitory effect on the proliferative activity of HaCaT cells (F =128.04,P ＜ 0.05),which significantly decreased along with the increase of UVB doses,with significant differences between the three UVB groups (P ＜ 0.05).Se-Met pretreatment also affected cellular proliferative activity (F =5.95,P ＜ 0.05),which was significantly increased in Se-Met (10 nmol/L-1 μmol/L) + UVB groups compared with the UVB groups at corresponding doses (all P ＜ 0.05).There was no significant interaction effect on cellular proliferative activity between UVB radiation and Se-Met pretreatment (F =1.65,P ＞ 0.05).The apoptosis rate of HaCaT cells in the 30-mJ/cm2 UVB group was 31.9％ ± 2.67％,significantly higher than that in the normal control group (4.1％ ± 0.67％,P＜ 0.05) and in the 10-,50-,100-,200-nmol/L and 1-μmol/L Se-Met + 30-mJ/cm2 UVB groups (21.9％ ± 3.72％,17.2％ ± 1.67％,4.6％ ±-0.85％,7.5％ ± 1.86％ and 13.5％ ± 1.95％ respectively,all P ＜ 0.05).Similarly,SOD and GSH-Px activities were significantly weaker (both P ＜ 0.05),while MDA levels were higher (all P ＜ 0.05) in the 30-mJ/cm2 UVB group than in the normal control group;however,there was a significant increase in SOD and GSH-Px activities but a decrease in MDA levels in the Se-Met (10 nmol/L-1 μmol/L) + 30-mJ/cm2 UVB groups compared with the 30-mJ/cm2 UVB group (all P ＜ 0.05).Conclusions Se-Met can reduce UVB-induced oxidative damage to HaCaT cells,likely by enhancing antioxidase activity and decreasing oxygen radicals.
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AIM: To investigate the protective effect of sodium selenite ( Na2 SeO3 ) on human keratinocytes under ultraviolet-B (UVB) irradiation.METHODS: The cultured HaCaT cells were divided into 4 groups: (1) normal control group;(2) Na2 SeO3 group:pretreated with Na2 SeO3 at doses of 10 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L and 1 μmol/L for 24 h;(3) UVB group: irradiated with UVB at doses of 300, 600 and 900 J/m2; (4) Na2SeO3 +UVB group:after pretreated with Na2SeO3 for 24 h, irradiated with UVB at doses of 300, 600 and 900 J/m2.The cell pro-liferation was detected by MTT assay .The apoptotic rates of HaCaT cells treated with UVB at dose of 300 J/m2 were as-sessed by flow cytometry .RESULTS:Compared with normal control group , the cell proliferation activity in UVB group de-creased significantly ( P<0.05 ) .The cell activity was inversely correlated with the irradiation intensity .No significant difference of the cell activity between Na 2 SeO3 group and normal control group was observed .The cell proliferation in Na2SeO3 +UVB group was higher than that in UVB group significantly (P<0.05).Na2SeO3 at concentration of 100 nmol/L showed the strongest activity to promote cell proliferation .After 300 J/m2 UVB irradiation, the apoptotic rate in Na2SeO3+UVB group decreased significantly ( P<0.05) compared with UVB group .The inhibitory effect of Na 2 SeO3 at concentra-tion of 100 nmol/L on apoptosis was the strongest .CONCLUSION: The damage of human keratinocytes by UVB irradia-tion is in a dose-dependent manner .The photoprotection performance of Na 2 SeO3 reduces the damage of human keratino-cytes induced by UVB irradiation .
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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.
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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.
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BACKGROUND: There are plenty of studies of estrogen effects on mammalian osteoblast, but the studies of estrogen effects on bird osteoblast cannot be found. There are many reports about the side effects of letrozole on bone metabolism, but there are no reports about the effect of letrozole on osteoblast.OBJECTIVE: The effects of estrogen and letrozole on the proliferation, cell cycle, estrogen receptor mRNA expression and alkaline phosphatase (ALP) activity of chicken osteoblast in vitro were studied in order to illustrate the mechanism of medullary bone osteogenesis.METHODS: The osteoblasts were harvested from the frontal bone of 15-day SPF chicken embryos by the enzyme digestion, and treated with various mass concentrations of estrogen (0, 5, 10, 20, 100. 200, 400,800, 2 000, 20 000 ng/L) and letrozole (0, 5.10, 25, 50, 100, 250, 500, 1 000, 5 000 μg/L). The proliferation rates of the osteoblast treated with estrogen or letrozole were measured through the MTT method, The ALP activities of osteoblast were measured by the pNPP method. The cell cycle was measured by flow cytometry. The expression of estrogen receptor mRNA was detected using real-time fluorescent quantitative polymerase chain reaction (PCR).RESULTS AND CONCLUSION: The estrogen could promote proliferation of osteoblast in concentration- and time-dependent fashion. Estrogen could increase the expression of estrogen receptor mRNA, impulse cell cycle, and elevate ALP activities.Letrozole could increase the cell population, impulse cell cycle, inhibit estrogen receptor mRNA expression, but letrozole has no effects on ALP synthesis and secretion.
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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.
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The present article reports the method of preparation of epidermal growth factor (EGF) from mouse submaxillary glands by using a two-step liquid chromatography and the efficacy of EGF in promoting animal wound healing. It was found that combined purification of crude EGF is better with Bio-gel p-10 and DEAE-DE52 ion-exchange chromatographies and purified EGF has a marked enhancement of wound healing and reduction of cicatricial contracture in animals.