Objective To study the effects of UVR or H2O2 on the expression of p53 in human melanocytes,and that of nutlin-3 and PFT-α on the DNA oxidative damage,and to investigate the role of p53 in the antioxidative stress.Methods The effect of UVR,H2O2,nutlin-3 and PFT-α on the expression of p53 of human melanocytes was detected by Western blot analysis,and that of nutlin-3 and PFT-α on UVR or H2O2 DNA damage assessed by single cell electrophoresis (comet assay).Determination of the effect of nutlin-3 on H2O2 DNA damage was detected by γ-H2AX immunofluorescence.Results UVR and H2O2 could induce p53 protein expression,accompanied by increased phosphorylation of p53 on serine 15 residue,and nutlin-3 and PFT-α could induce and inhibit p53 protein in human melanocytes respectively; nutlin-3 decreased the tail moment of DNA oxidative damage of UVR or H2O2 in human melanocytes,but PFT-α increased the tail moment of DNA oxidative damage of UVR or H2O2 in human melanocytes,and there were significant differences among the control and exposed groups; nutlin-3 decreased expression of γ-H2AX.Conclusions p53 plays a very important role in the antioxidative stress in melanocyte exposed to UV or H2O2.

Objective To estimate the effect of a cis-imidazoline derivative,nutlin-3,on the biological behavior of A375 human melanoma cells,and to investigate its mechanism.Methods Cultured A375 cells were divided into several test groups treated with nutlin-3 at different concentrations (2.5,5,10 μmol/L) for 24,48 and 72 hours,and a control group treated with dimethyl sulfoxide (DMSO) only.Then,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,Western blot to measure the expression of p53 protein,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,and Transwell assay to evaluate migratory activity,of A375 cells.Statistical analysis was carried out by repeated-measures analysis of variance (ANOVA).Results After treatment with nutlin-3 of 2.5,5 and 10 μmol/L for 24,48 and 72 hours,significant differences were observed among different time points at each concentration and among different concentrations at the same time point in proliferation inhibition rate (F =67.43,135.58,respectively,both P ＜ 0.01),p53 protein expression level (F =1255.00,9196.00,respectively,both P ＜ 0.01),percentage of cells at G2 phase (F =831.38,267.99,respectively,both P ＜ 0.01),apoptosis rate (F =809.45,723.83,respectively,both P ＜ 0.01),migration inhibition rate (F =1100.00,1667.00,respectively,both P ＜ 0.01).The influence of nutlin-3 on cellular proliferative activity increased with the increase in its concentration,and that on percentage of cells at G2 phase,apoptosis rate and migratory activity increased with the increase in its concentration and treatment duration.There was a significant interaction between the treatment duration and concentration of nutlin-3 for p53 protein expression level in (F =826.79,P ＜ 0.01),percentage of cells at G2 phase in (F =21.602,P ＜ 0.01),apoptosis rate in (F =44.48,P ＜ 0.01),migratory activity of (F =313.09,P ＜ 0.01),and cellular proliferative activity of (F =26.95,P ＜ 0.01),A375 cells.Conclusion Nutlin-3 may inhibit the proliferation and migration of,but promote cell cycle arrest and apoptosis in,A375 cells,through accumulation of p53 protein.

ObjectiveTo evaluate the effects of all-trans retinoic acid(ATRA) on melanin content,activity and protein expression of tyrosinase,mRNA expression of Cu/Zn superoxide dismutase(SOD) in A375 cells irradiated with ultraviolet B(UVB).MethodsCultured A375 cells were classified into 6 groups:ATRA+UVB group treated with ATRA after UVB irradiation,hydroquinone+UVB group treated with hydroquinone after UVB irradiation,UVB group and ATRA group treated with UVB irradiation and ATRA respectively,negative control group receiving no treatment.Melanin content and tyrosinase activity were determined by NaOH solubilization assay and dopa-oxidation assay respectively at 24,48 and 72 hours after the addition of ATRA into medium.Western blot was performed to detect the protein expression of tyrosinase,and real-time quantitative PCR to measure the mRNA expressions of tyrosinase and Cu/Zn SOD in A375 cells after 24-hour culture with ATRA.ResultsThe melanin content and tyrosinase activity decreased in UVB-irradiated cells after being treated with ATRA for 24,48 and 72 hours.The protein (gray scale) and mRNA (2-△△Ct value) expression levels of tyrosinase were 0.72 ± 0.070 and 1.400 ± 0.135 respectively at 24 hours after UVB irradiation,decreased to 0.42 ± 0.056(P ＜0.01) and 0.810 ± 0.062(P ＜ 0.01 ) respectively after additional treatment with ATRA.The mRNA expression level of Cu/Zn SOD was 0.323 ± 0.066 in A375 cells at 24 hours after UVB irradiation,and increased to 0.625 ±0.103 (P ＜ 0.01 ) after additional treatment with ATRA.ConclusionATRA can suppress UVB-induced increase in melanin synthesis and elevate Cu/Zn SOD level in A375 cells,likely through tyrosinase pathway.

Objective:To generate rabbit-anti B16 melanocyte polyclonal antibodies and study the effect on melanocyte proliferation.Methods:Rabbits was immunized with B16 melanocytes to produce rabbit anti-B16 melanocyte polyclonal antibody(pAb).The titer of the antiserum was then detected using the tube agglutination assay;The antiserum was purified through the G protein affinity chromatograph,and the molecular weight of the purified Ab was identifed through SDS-PAGE.The effect of purified IgG on B16 melanocytes proliferation was detected using MTT assay.Results:The titer of the antiserum reaches 1∶1 280;SDS-PAGE shows that the heavy chain molecular weight of the purified IgG is 66.2 kD;MTT assay shows that the IgG fraction inhibited the proliferation of B16 melanocytes with signifancent difference when compared to none IgG or no purified antiserum.Conclusion:The rabbit anti-B16 melanocytes pAb with high titer is preparated successfully.The purified IgG can inhibit proliferation of B16 melanocytes.It could be useful in studying the effect of this antibody on melanocyte growth and pigment metabolism,it also be useful in studying the pathogenesis of vitiligo.

Objective To investigate the efficacy of recombinent human B-type natriuretic peptide (rhBNP) in peripartum cardiomyopathy (PPCM) with refractory heart failure (RHF).Methods From January 2010 to January 2014,a total of 61 patients with PPCM-RHF of the First Hospital of Qinhuangdao were enrolled into this study.All patients were randomly assigned into the rhBNP group (30 cases) and control group (31 cases).rhBNP was given 1.5 μg/kg,2 mim intravenously in rhBNP group and then infused intravenously 0.007 5-0.030 μg/(kg · min) for 24 hours,0.015-0.030 μg/(kg · min) for 24 hours when systolic blood pressure(SBP) ≥ 85 mmHg (1 mmHg =0.133 kPa) and mean brachial arterial pressure (MBP) ≥ 65 mmHg.Routine therapy followed the clinic practice was administrated in control group for 24 hours.Recorded the dyspnea change.Blood samples were collected at different time points to investigate BNP at 6 h,14 d and 30 d.Ultrasonic cardiography (UCG) was performed to evaluate left ventricular ejection fraction (LVEF) and left ventricular end-diastolic dimension(LVEDD) before the initiation of the infusion,at day 1,14 and 30 after the infusion.All patients were followed up for one month to record the main adverse cardiac events (MACE),including heart failure recurrence,cardiac death and ventricular fibrillation and tachycardia.Results The time of dyspnea resolved in rhBNP group was significantly shorter than control group ((1.69 ± 1.07) h vs (2.69 ±1.39) h,P =0.002).Concentration of plasma BNP(6 h:(296.50±123.25) ng/L,14 d:(141.37±69.54) ng/L,30 d:(107.41±33.69) ng/L) was signifisantly reduced in both groups than basic line (727.07 =± 146.84) ng/L,and it was significantly different between two groups.LVEF raised and LVEDD decreased were observed at 24 hours((52.23±4.54) mm),14 d((49.60±4.20) mm) and 30 d((42.59±3.90) mm) in rhBNP group and were significant better than the control group ((56.33 ± 4.38) mm,(53.03 ± 4.95) mm,(48.85±4.96) mm;P ＜0.05).There was no significant difference in term of LVEF between two groups after treatment.However,24 h(4.12±41.13)％,14 d(4.10±43)％,30 d(44.52±3.43)％ were significantly higher than the baseline(36.73±5.82)％ in rhBNP group.MACEs were lower in rhBNP group at 30 dayscontrast to the control(10％ (3/30) vs.42％ (13/31);P =0.005).Conclusion Compared with conventional treatment,rhBNP can effectively improve the heart function in patients with PPCM-RHF,reduce the occurrence of major adverse cardiac events in 30 d,and improve the prognosis of patients.

Objective To report and analyze the renal function improvement in a case with ad-vanced bilateral renal cell carcinoma after targeted therapy. Methods The patient was a 60-year-old man who complained of lower back pain for 1 month. Ultrasound and CT scan detected bilateral renal masses, left lesion was 11.0 cm×9.4 cm×8.5 cm, and the right one was 3.5 cm×4.3 cm×4.1 cm. X-ray examination showed metastatic lesions in liver and lower right lung. GFR was 20.39 ml/min of left kidney, 25.40 ml/min of right kidney. The renal biopsy confirmed renal clear cell carcinoma. Sorafenib was administrated 400 mg twice or once daily for 12 weeks. Results After the targeted therapy, the decreased bilateral kidney tumor sizes were identified by CT scan. There was liquid nec-rosis in the tumor, and no new metastatic lesion detected. The kidney function was improved as well. The total GFR increased to 71.38 ml/min. Left kidney GFR increased to 31.57 ml/min, right kidney GFR increased to 39.81 ml/min, respectively. Conclusion Targeted therapy could improve renal function in advanced renal cell carcinoma cases by controlling tumor development.

By skin prick test,three kinds of mite allergens(Dermatophagoides pteronyssinus,Dermatophagoides farinae,Blomia tropicalis)were tested in a group of asthma children in Jinhua area from Oct 2005 to Sep 2006.The pos-itive rate to allergen from D.pteronyssinus and D.farinae was 80.6% and 77.8% respectively,higher than that of Blomia tropicalis(61.1%)(?2=21.39,P

Shennongjia is well known as the national treasure of traditional Chinese medicine resources at home and abroad. In order to provide Shennongjia with better protection of traditional Chinese medicine and promote the sustainable utilization of its resources, based on the specific analysis of She nnongj ia′s conservation status and
available resources, we put forward primary strategies and specific measures for the sustainable utilization framework of Shennongjia traditional Chinese medicine resources, in view of resources conservation, development, utilization, as well as the resources administration.

To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.

Objective To discuss the clinical characteristics of adrenal metastases,and summarize the experience of diagnosis and treatment.Methods From January 2008 to June 2018,the clinical data of 55 patients with adrenal metastases treated in our hospital were analyzed retrospectively.This study included 34 male patients and 21 female patients and the median age was 60 years old (ranged 55 to 84 years old).The median value of maximum diameter of adrenal metastases was 3 cm (ranged 1.9 to 10.3 cm);with 35 cases on the left side,13 cases right and 7 cases bilateral.The primary sites of malignant tumors were pancreas (18 cases,32.7％),lung (12 cases,21.8％),liver (6 cases,10.9％) and colorectum (6 cases,10.9％),respectively.Thirty-four cases were confirmed by pathology after adrenalectomy and 21 cases were confirmed by needle biopsy.Thirty cases were diagnosed synchronously with the primary tumor and 25 cases were metachronous.The median time from diagnosis of primary tumors was 13.3 months (ranged 2.0 to 97.4 months).42 cases of these 55 cases were diagnosed within one year.Treatment options for adrenal metastatic lesions included single adrenalectomy in 18 cases,adrenalectomy combined with radiotherapy 16 cases,single intravenous chemotherapy 10 cases,intravenous chemotherapy combined with non-operative treatment 10 cases and single radiotherapy 1 case.Results The main pathological types were adenocarcinoma (19 cases,34.5％),ductal adenocarcinoma (10 cases,18.2％),hepatocellular carcinoma (6 cases,10.9％) and clear cell carcinoma (4 cases,7.3％).Two cases were lost follow-up and the follow-up rate was 96％,the median follow-up time was 8 months (ranged 1 to 135 months).The median overall survival (OS) time of 55 patients was 5.3 months (ranged 1 to 134 months).The one-year survival rate was 23.6％ (13/55),the two-year survival rate was 12.7％ (7/55),the three-year survival rate was 9.1％ (5/55) and the five-year survival rate was 1.8％ (1/55).Conclusions Pancreatic cancer was the most common type of malignant tumor for adrenal metastases in our hospital.Most primary tumors and adrenal metastases were diagnosed synchronously or within one year.Comprehensive treatment with retroperitoneal adrenalectomy may improve the OS,however the overall prognosis is poor.