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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.
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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.
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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.
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Objective To survey the prevalence, distribution of non-motor symptoms (NMS) in essential tremor (ET) and the relationship with disease severity and duration.Methods Fahn-Tolosa-Matin Tremor Rating Scale (TRS) was used to assess motor symptoms in 62 patients with ET.The Parkinson's disease (PD) NMS Questionnaire and T&T olfacmeter and Mini-mental State Examination (MMSE) were used to explore non-motor symptoms in ET patients.Results In ET, a range of NMS occurred across all disease stages.More than half patients (51.6%, 32/62) had olfactory dysfunction,significantly higher than the healthy control group (30.0%, 18/60, x~2=12.371, P<0.05).A third had hyposmia.16.1% had partial olfactory loss.Each ET patient had 5 different NMS on average.Seven NMS were more common in ET patients than in control, including remembering, olfactory dysfunction, intense vivid dreams, anhedonia, depression, anxiety, sleep disorders.The incidences of remembering, olfactory dysfunction,intense vivid dreams were 58.1% (36/62),51.6% (32/62),48.4% (30/62), ranked top 3 in ET patients.Olfaction had inverse correlation with age, while there was a negative correlation between NMS score and TRS score, gender, disease duration and weather to be treated.Conclusion Besides posture tremor and kinetic tremor,NMS occur in ET,and should be well recognized and treated.
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Objective To study the incidence of depression in essential tremor (ET) and associated factors. Methods Depression in 62 ET patients and 60 healthy subjects as control was evaluated by means of Hamilton Depression Scale ( HAMD ) , as well as Fahn-Tolosa-Marin Tremor Rating Scale (TRS) and Pittsburgh Sleep Quality Index (PSQI). Results Fifty-three point two percent(33/62) of ET patients and 11.7% (7/60) of healthy subjects were found to have at least mildly depression (HAMD score of 8 or higher), 35. 5% (22/62) of ET patients and 8. 3% (5/60) of healthy subjects fell into the mildly-to-mederately depression (HAMD score between 8 and 20 ), 17.7% (11/62) of ET patients and 3.3% (2/60) of healthy subjects were classified into moderately-to-severely depressed range (HAMD score between 21 and 35). There were statistical differences in ET group and healthy subjects group (X2= 23.898, 13.043, 6.649, all P <0.01). Additionally, there were statistical differences in anxiety/ somatization (t=-6.747, P<0.01), cognitive impairment (t=-2.017, P=0.05), block(t= -4.145, P<0.01), sleep disorders (t=-4.500, P<0.01) and despair (t=-3.591, P<0.01) between depression group and non-depression group. There were marked differences in PSQI total score ( t =-3.196, P=0.003 ), subjective sleep ( F1, t=-3.037, P=0.004), quality sleep latency (F2, t= -4.674, P<0.01) and sleep disturbances (F5, t=-2.594, P=0.013 ) between depression disorder group and non-depression disorder group. Meanwhile, the score of TRS, PSQI and sex were closely correlated with HAMD (β=0.589, P=0.000 ;β=0.469,P=0.000 ;β=0.256, P=0.027 ). Conclusions The incidence of depression is high in ET. Manifestation of depression are anxiety, reduced interest in work, sleep disorders, retardneas, inferiority complex, etc. The degree of symptoms relates to the severity of ET, sleep quality and gender.
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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
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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.