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.

Informed consent right is not just for basic ethical consideration, but is important for protect-ing patient’s right by law, which is expressed through informed consent contract. The appraised individ-uals of forensic clinical examination have the similar legal status as the patients in medical system. However, the law does not require informed consent right for the appraised individuals. I recommend giving certain informed consent right to the appraised individuals in the forensic clinical examination. Under the contracted relationship with the institution, the appraised individuals could participate in the examination process, know the necessary information, and make a selected consent on the examination results, which can assure the justice and fairness of judicial examination procedure.

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 investigate the influence of cognitive behavioral intervention on self efficacy and quality of life in patients with chronic hepatitis B (CHB).Methods 90 patients with chronic hepatitis B were randomly divided into cognitive behavioral intervention group and control group,45 cases in each group.The patients of the control group were treated with routine treatment,the intervention group was given cognitive behavior intervention on the basis of conventional treatment.The general self efficacy scale (GSEs) and SF-36 quantity form (SF-36) were used to evaluate the effects of intervention.Results Before the intervention,the GSES score and SF-36 scores of each dimension of the two groups had no statistically significant differences (all P ＞ 0.05).After intervention,the GSES scores of the intervention group was (22.53 ± 4.12) points,which was significantly higher than (17.82 ± 4.51)points of the control group,there was significant difference between the two groups (t =4.918,P ＜0.01).The dimensions of the intervention group SF-36 score[comprehensive health status (16.13 ± 2.04)points and physical function (17.73 ± 3.49) points,mental health (15.73 ± 2.69) points,role limitations (18.38 ± 2.78) points,social function (14.76 ± 2.96) points,physiological (15.89 ± 2.85) points,vitality and energy (19.18 ± 3.43) points,body pain (19.84 ± 3.78) points] were significantly increased,and compared with the control group [general health status (12.62 ± 2.15) points and physical function (13.18 ± 2.31) points,mental health (9.24 ± 3.54) points,role limitations (8.67 ± 3.47) points,social function (9.24 ± 2.42) points,physiological (8.67 ± 2.60) points,vitality and energy (10.64 ± 2.73) points,body pain (10.80 ± 2.40) points],the differences were statistically significant (t=6.896,8.863,9.189,17.309,9.287,12.046,11.645,14.937,all P＜0.05).Conclusion Cognitive behavioral intervention can significantly enhance the self-efficacy of patients with hepatitis B,and improve the quality of life of patients,it can be popularized in clinical work.

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 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 investigate the expression of p38 mitogen actirated protein kinase (p38MAPK) in renal fibrosis in diabetic mice. Methods Male homozygous C57 BL/6 mice were divided at random into control group intraperitoneally(i. p.) injected with citrate buffer and diabetes group received 5 consecutive daily intraperitoneal inserum glucose level exceeding 16.7 mmol/L. Mice were killed at 0,4weeks ,8weeks, 12weeks and 16weeks respectively after STZ injected. The kidney tissues were obtained from diabetic and control mice. Serum glucose, extracellular matrix,and 24 h urinary albumin excretion rate(UAE)and the serum creatinine(Scr) were measured. The kidney tissue was used for histological and morphometric studies of glomerular matrix expansion (PAS), and the expression of phosphorylated p38MAPK and TGFβ1 were measured by immunohisteehemical staining. p38MAPK and TGFβ1mRNA were analyzed by reverse transeriptase-PCR. Results The serum level of glucose in diabetic mice increased significantly. Scr and 24 h UAE, and glomerular matrix expansion in diabetic mice were obviously higher than that in control mice. Phosphorylated p38MAPK and TGFβ1 levels were obviously increased in the kidney of diabetic mice compared with those in control mice(P ＜0. 01) ;TGFβ1 expression was positively related to the expression of phosphorylated p38MAPK. Conclusion The overexpression of phosphorylated p38MAPK in kidney shoud play an important role in the development of renal fibrosis associated with diabetic nephropathy in mice.

AIM:To investigate the expression and probable role of extracellular signal-regulated protein kinase(ERK1/2)in renal fibrosis associated with diabetic in mice.METHODS:Male homozygous C57BL/6 mice were divided at random into control group(intraperitoneally injected with citrate buffer)and diabetes group(received 5 consecutive daily intraperitoneal injections of streptozotocin at dose of 50 mg?kg-1?d-1).All mice were followed up for 16 weeks.Diabetes was confirmed by serum glucose levels exceeding 16.7 mmol/L.Mice were killed at 0,4,8,12 and 16 weeks respectively after streptozotocin injection.The kidney tissues were obtained from diabetic and control mice.Serum glucose,kidney weight/body weight(KW/BW),24 h albumin excretion rate(UAE)and the serum creatinine(Scr)were measured.The kidney tissue was used for histological and morphometric studies of glomerular size,glomerular matrix expansion(PAS),and the expression of TGF-?1,phosphorylated ERK1/2 and collagen Ⅲ by immunohistochemical staining.RESULTS:The serum level of glucose in streptozotocin-induced diabetic mice increased significantly.The kidney weight/body weight ratio,glomerular volume and glomerular matrix expansion in diabetic mice were obviously higher than those in control mice.Serum creatinine and 24 h albumin excretion rate in diabetic mice increased significantly compared with control mice.TGF-?1,phosphorylated ERK1/2 and collagen Ⅲ levels were obviously increased in the kidney of diabetic mice compared with those in control mice(P

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.

AIM: To investigate the functional role of TGF-?_1 signal protein Smad2/3 in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats. METHODS: The unilateral ureteral obstruction (UUO) model was induced by the ligation of left ureter. Rats were sacrificed at 1, 3, 7, 14, 21, and 28 days after UUO was initiated. TGF?_1 protein, phosphorylated Smad2/3 and interstitial ?-smooth muscle actin (?-SMA) expression were assayed by immunohistochemical staining. TGF-?_1 mRNA in the obstructed kidney was analyzed with in situ hybridration. HE and Masson staining were used for histological and morphometric studies of the pathological change in obstructed kidney. RESULTS: The results showed that upregulation of TGF-?_1 in tubulointerstitium of both cortex and medulla at day 3 (a 3.1 fold increase vs control, P