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.

Pituitary adenoma, a benign intracranial tumor, is the third most common brain tumor, second only to glioma and me-ningioma. Pituitary adenoma has been defined as a benign tumor, but some pituitary adenomas can invade the surrounding tissue. This tumor is difficult to resect and can easily recur after surgery. RWD-containing sumoylation enhancer (RSUME) can stabilize the activity of hypoxia-inducible factor-1α and inhibitor kappaB by the small ubiquitin-related modifiers. This phenomenon indicates the impor-tance of RSUME in pituitary adenoma because it promotes the invasiveness of the tumor. However, the correlation between RSUME and the invasion of pituitary adenoma remains unclear. In this study, the roles of RSUME on HIF-1α/VEGF signaling pathway and IκB/NF-κB compomers in the invasiveness of pituitary adenoma were reviewed.

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 establish a novel rapid detection method based on PCR-single-strand-conformational polymorphism (PCR-SSCP) to determine mutation of streptomycin-resistance associated rpsL and rrs genes in isolates of Mycobacterium tuberculosis (MTB).MethodsStreptomycin-resistance of 112 MTB isolates was detected using the routine drug susceptibility test,and a special PCR-SSCP assay was established.The mutations of rpsL and rrs genes in streptomycin-resistant MTB isolates were detected by PCR-SSCP and PCR direct sequencing (PCR-DS) ; the results from two techniques were compared.Results All isolates had both rpsL and rrs genes.Fifty-two isolates (46.4％) were streptomycin susceptible,in which only 1 isolate showed abnormal PCR-SSCP fragments from rrs gene,and the specificity of PCR-SSCP was 98.1％ (51/52).Sixty isolates (53.6％) were streptomycin-resistant,in which 46 (76.6％) and 11 ( 18.3％ ) isolates presented the abnormal PCR-SSCP fragments of rpsL and rrs gene,respectively.One streptomycin-resistant isolate showed abnormal PCR-SSCP fragments from both rpsL and rrs genes.The sensitivity of PCR-SSCP was 93.3％ (56/60).ConclusionThe PCR-SSCP that established in this study is a specific and sensitive method for rapid detection of the streptomycin-resistance associated mutations in rpsL and rrs genes of MTB.

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.

BACKGROUND: Studies have demonstrated that the freeze-dried and irradiation-sterilized allogeneic bone is an ideal material for bone transplantation, they are present with good biocompatibility and biomechanical property, also maintains some necessary enzymes for bone morphogenetic protein and morphogenesis protein in bone matrix with some osteninductivable potentials. OBJECTIVE: To observe the effect of the compound of placenta tissue injection and allogeneic lyophilized bone on the reconstruction of jaw bone defects of dogs, and to compare with single allogeneic lyophilized bone. DESIGN, TIME AND SETTING: A comparative observational trial was performed in the Animal Experimental Center of Harbin Medical University between December 2007 and September 2008. MATERIALS: Eight healthy hybred adult dogs; allogeneic lyophUized bone was offered by Hubei Osteolink Biomatedals Co.,Ltd; placenta tissue injection was purchased from Livzon Pharmaceutical Factory Zhuhai (2 mL per injection); allogeneic lyophilized bone: placenta tissue injection=(4-5):1.METHODS: A total of 96 experiment areas from hemisphere jaw defect models at 1.0 cm diameter were established in dog jaw bone site corresponding with central incisor, canine teeth and root apex of the first molar. In the experiment group, the allogeneic lyophilized bone and bone particles were soaked in placenta tissue injection and under saturation state, then the compound of placenta tissue injection and allogeneic lyophilized bone were implanted to jaw bone defect. In the positive control group, the allogeneic lyophilized bone and bone particles were soaked in sodium chloride injection and under saturation state, then implanted to jaw bone defect. In the negative control group, nothing was implanted to jaw bone defect. Each experiment area comprised four materials in each group.MAIN OUTCOME MEASURES: The radiological and histological observations were performed at 2, 4, 8 and 12 weeks after operation.RESULTS: In the experiment group, there was obvious cartilaginous osteogenesis in the earlier period and intramombranous osteogenesis in the late period. The new bone was well integrated with the surrounding tissues. In the positive control group, new recovered bone existed but the combination between the new bone and the original bone was not well. In the negative control group, jaw bone defects were not filled with bone trabecula. Histological examination results showed that there were more new bones in the experiment group than the control groups at 2, 4, 8 and 12 weeks after operation. Statistical difference could be observed among them (P < 0.05, P < 0.01 ).CONCLUSION: The compound of placenta tissue injection and allogenalc lyophilized bone can promote recovery of jaw bone defect actively and shorten recovering time effectively.

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.

ObjectiveTo understand the feature of blood pressure in children aged 7 to 12 years in Zhengzhou.Methods According to stratiifed cluster random sampling method, children aged 7 to 12 years in ifve schools from three urban and two suburban counties in Zhengzhou were analyzed. The height, weight, waist circumference, hip circumference, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured and analyzed.Results The survey included total 6460 children aged 7 to 12 years, 3206 urban children (49.63%), 3254 suburban children (50.37%), 3525 boys (54.57%) and 2935 girls (45.43%). SBP in boys [(117.86±18.18) mmHg] was signiifcantly higher than that in girls [(113.82±13.11) mmHg (t=3.16;P=0.002). The incidence of hypertension in children in Zhengzhou was 7.52%. The blood pressure in boys was higher than that in girls (χ2=9.66,P=0.002). The blood pressure in urban boys and girls was higher than that in suburban boys and girls respectively (χ2=24.15, 14.39;P=0.000). The SBP and DBP had positive correlation with age, height, weight, BMI, waist circumference in boys (P<0.01). The SBP had positive correlation with age, height, weight, BMI, waist circumference in girls (P<0.01). Conclusions The blood pressure is higher in boys than in girls, which also is higher in urban children than in suburban children in Zhengzhou. The SBP is related to the age, height, weight, BMI, waist circumference.

Objective To explore the imaging characteristics of testicular germ cell tumors and to improve the MRI diagnostic level. Methods MRI and clinical data of 25 cases confirmed testicular germ cell tumor by pathological examination were retrospectively analyzed. All the 25 cases were performed plain scan of MRI,and 16 patients underwent MRI enhanced scan.The size,morphology,signal intensity, adjacent structures,enhancement figure and tumor supplying artery were assessed and the histopathological findings were servered as the standard of reference.Results In the all 25 testicular germ cell tumors,10 cases were seminoma,8 cases showed homogeneous low signal intensity,2 cases of seminoma were low signal intensity on T2 WI,furthermore 5 cases performed poor nodular enhance-ment,2 cases performed homogeneous enhancement,4 cases performed fibrous septa enhancement.4 cases were yolk sac tumor ap-peared equal-low signal on T1 WI,slightly high signal intensity on T2 WI and progressive enhancement.Mature teratoma,pidermoid cyst and mixed germ cell tumor were 3 cases respectively,the MRI demonstrated mixed low signal intensity on T1 WI and mixed high signal on T2 WI.2 cases were embryonal carcinoma demonstrated middle-low signal intensity on T1 WI,and mixed low signal intensity on T2 WI.The two cases revealed bleeding signal intensity and septa enhancement.Conclusion MRI can be used to diagnose germ cell tumors with high accuracy,and provides essential information for pathological type,stage and differential diagnosis.

ObjectiveTo investigate the influence of TGF-β receptor subtypes expression and their downstream signaling Smad proteins on rat renal interstitial fibrosis induced by unilateral ureteral obstruction(UUO).MethodsA total of 90 rats were randomly divided into three groups:normal control(CON),sham operation (SOR) and UUO group,and sacrificed 1,3,7,14 and 21 days after operation.Serum creatinine and urea nitrogen were detected to assess renal function.PAS and Masson staining were performed to observe histological damage in the kidneys.Quantitative RT-PCR was used to define expression of mRNA encoding TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney tubular cells.Real-time PCR,Western blotting and immunofluorescence were used to monitor the time-related expression of the TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney.ResultsCompared with the CON group,serum creatinine and urea nitrogen in UUO groups increased at day 3 after operation (P＜0.05) and reached their peak 21 days after operation (P＜0.01).Obvious inflammatory cell infiltration was observed in UUO group 3 days after operation,while renal tubular atrophy and renal interstitial fibrosis were observed in UUO group14 days after operation.The mRNA expressions of ALK-5,ALK-7 and TGF-βR Ⅱ increased significantly in UUO group 3 days after operation (all P＜0.05) and reached their peaks 14 days after operation (all P＜0.01).The mRNA expression of ALK-6 decreased significantly in UUO group 3 days after operation(P＜0.05) and reached its lowest level 14 days after operation (P＜0.01).The changes in the protein level of those receptors were consistent with their mRNA expressions.The protein expressions of Smad2/3 and p-Smad2/3 increased significantly in UUO group at day 3(all P＜0.05) and reached their peak at day 14 after operation(all P＜0.01).ConclusionExpressions of TGF-β receptor subtypes ALK-5,ALK-6,ALK-7,TGF-βR Ⅱ and their downstream signaling Smad2 and Smad3 proteins may influence the progress of renal interstitial fibrosis,tubular atrophy and inflammatory cell infiltration in UUO model rats.