There is great progress in the study of the diagnosis of hyperkinetic disorders in China within the latest ten years, but it is not enough. Three features are referred by reanalyzing 281 articles dating from 1997 to 2004 collected by the CJFD: 1) Several editions of diagnostic criteria and diagnostic methods are simultaneously used; 2) Comparing with the rate of utilization of the new edition, the old editions of diagnostic criteria constitute a small percentage ; 3) Diagnostic criteria and diagnostic tools have some shortcomings. Four recommendations maybe help to solve these problems:1) Make parents and teachers advance their acknowledgement of hyperkinetic disorders; 2) Develop and improve the diagnostic criteria and diagnostic tools through more researches; 3) Use advanced scientific apparatuses more widely; 4) Establish a perfect diagnostic system according to our culture.

The muscle tonus of 7 (43 trials) swimmers were measured by the side of theswimming pool before and after training when they felt muscle stiffness and sore-ness. The muscle tonus and EMG of 10 (20 trails) swimmers and 5 (10 trails) runnrswere also measured and taken in the laboratory when they had a pain in the muscleafter training. The results showed that after training the muscle tonus was significantly higherthan before and the athletes had muscle stiffness and soreness. After stretching,not only the muscle tonus decreased, the athletes also felt better. The amplitude of the EMG appeared higher in the first stretching. then it be-came lower and lower with the stretching even showed electric silence. At the sametime, the muscle tonus decreased and the soreness was relieved. This study suggested that the early muscle soreness and stiffness are not cau-sed by local tissue edema, which is thought to be due to biochemical end-productsof metabolism, especially lactic acid. If muscle soreness and stiffness after vigo-rous exercises are the result of tissue edema, they can not be readily relieved byway of stretching technique since the water causing tissue edema cannot be remo-ved from the muscle tissue to the circulation in only about 30 seconds. Therefore, in our opinion, the early muscle soreness and stiffness are not cau-sed by biochemical but by physiological reasons, i. e. the physiological reflex spasmrelated with the functional state of the muscle spindle sensing system. Of course.this still needs to be confirmed by more extensive study in the future.

Objective To investigate the changes in cathepsin B (CatB) expression in acutely photodamaged human dermal fibroblasts (HDFs) and their significance.Methods HDFs were isolated from the foreskin of children,and subjected to primary culture and subculture.The fourth-to eighth-passage HDFs were used in the following experiment.HDFs were divided into two groups to receive irradiation with different doses of ultraviolet A (UVA) for different durations (acutely photodamaged group) or remain unirradiated (control group).Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferative activity of HDFs after irradiation with UVA at 5,10,15,20 and 25 J/cm2 respectively.Western blot and quantitative real-time reverse transcription PCR were performed to measure the protein and mRNA expressions of CatB respectively in HDFs at 24,48 and 72 hours after exposure to UVA at 10 J/cm2,and at 48 hours after exposure to UVA at 10,15,20 and 25 J/cm2.Statistical analysis was carried out by analysis of variance and least significant difference (LSD) test using the SPSS 13.0 software.Results UVA radiation induced a decrease in the proliferative activity of HDFs.When the dose of UVA was ≤ 10 J/cm2,the survival rate of HDFs maintained higher than 85％,and significant differences were observed in cell survival rate between unirradiated and irradiated HDFs at 24,48 and 72 hours (all P ＜ 0.05).Western blot showed that the gray value of CatB protein in the acutely photodamaged group irradiated with 10 J/cm2 UVA was significantly higher than that in the control group at 24 hours (0.76 ± 0.14 vs.0.35 ± 0.01,P ＜ 0.05),48 hours (1.34 ± 0.38 vs.0.45 ± 0.12,P＜ 0.05) and 72 hours (0.82 ± 0.09 vs.0.61 ± 0.06,P＜ 0.05).Increased mRNA expressions of CatB were also observed in the acutely photodamaged group compared with the control group at 24 hours (0.149 ± 0.009 vs.0.089 ± 0.015,P ＜ 0.05),48 hous (0.173 ± 0.009 vs.0.091 ± 0.010,P ＜ 0.05) and 72 hours (0.185 ± 0.158 vs.0.111 ± 0.017,P ＜ 0.05) after UVA radiation at 10 J/cm2.The gray value of CatB protein was 0.99 ± 0.07,1.49 ± 0.14,1.89 ± 0.08,2.07 ± 0.06 in HDFs at 48 hours after exposure to UVA of 10,15,20 and 25 J/cm2,respectively,significantly higher than that in the control group (0.60 ± 0.05,all P ＜ 0.05).Similarly,the mRNA expression of CatB was up-regulated in HDFs at 48 hours after UVA radiation at 10,15,20 and 25 J/cm2 compared with the unirradiated HDFs.Conclusion The protein and mRNA expressions of CatB are up-regulated in acutely photodamaged HDFs induced by UVA radiation.

Objective To explore the inhibitory effect of harmine on melanoma A375 cells and its mechanism thereof.Methods (1) Melanoma A375 cells were treated with harmine at 0,0.5,1,2,5,10,20,50 and 100 mg/L for 48 h in vitro.CCK-8 method was used to detect the cell viability and confirm the experimental concentrations.(2) After the cells were treated with 0,1,2 mg/L harmine,the scratch and transwell assays were used to detect the cell migration and invasion ability.Western blot assay was used to detect the expression levels of epithelial mesenchymal transition (EMT)-related protein E-cadherin,N-cadherin,Snail and p53.(3) Three groups of ceils were set up.The control group was transfected with empty vector ordy.The empty vector group was transfected with empty vector after treated with 2 mg/L harmine for 24 h.The Snail transfection group was transfected with Snail cDNA after treated with 2 mg/L harmine for 24 h.The cell migration and invasion ability were detected after the transfection.Results (1) When the concentration of harmine was above 2 mg/L,the survival rate of A375 cells was significantly lower than that of the control group with the increase of harmine concentrations (P ＜ 0.05).Then,the concentrations of 0,1 and 2 mg/L of harmine were used in the following experiments.(2) With the increase of the harmine concentrations,the number of cells in the scratched area and the number of trans-membrane cells in each group were significantly decreased.The migration and invasion ability of the ceils were decreased gradually.The expression levels of E-cadherin and p53 were increased,while the expression levels of N-cadherin and Snail were decreased.(3) Cell transfection experiments showed that the migration and invasion ability of the cells were increased compared with those of empty vector group after transfection with Snail.Conclusion Harmine can inhibit the proliferation of A375 cells and decrease the abilities of metastasis and invasion,which may be achieved by decreasing the expression of Snail after activating the p53,thereby increasing E-cadherin and down-regulating N-cadherin to inhibit the EMT process.

OBJECTIVE　To develop an asssay for the quantitative determination of phillyrin in Shuanghuanglian tablet.METHODS　TLCS method was selected to determine the content.RESULTS　The linearity was obtained over the range of 0.31～ 1.55 μg(r=0.9996).The average recovery was 96.9% with RSD=1.49%.CONCLUSION　The results showed that this method is sensitive,simple,specific and accurate for the determination of tetrahydropalmatine in Shuang Huanglian tablet.

Objective To investigate the expressions of Th17, CD4 +CD25 +Treg, HLA-DR mRNA in peripheral blood of children with EV71-induced hand, foot and mouth diseases ( HFMD ) and their clinical significance.Methods Stratified random sampling was used to select 60 children with HFMDs from Liaocheng People’s Hospital from February to October, 2014, including 20 mild, 20 severe and 20 critically ill cases.Twenty healthy children were also enrolled as the control group.All the children with HFMDs were given ribavirin (10 mg/kg) for the treatment.The percentages of Th17 and CD4 +CD25 +Treg cells in CD4 +T cells of peripheral blood were determined by flow cytometry, and the expression of HLA-DR mRNA in peripheral blood mononuclear cells was detected by reverse transcription polymerase chain reaction ( RT-PCR).Analysis of variance and SNK-q test were used to compare the expressions of Th17, CD4 +CD25 +Treg and HLA-DR mRNA among groups, and Pearson correlation analysis was performed to reveal the correlations between HLA-DR mRNA and Th17, CD4 +CD25 +Treg.Results Compared with the control group, the expression of Th17 was increased, while CD4 +CD25+Treg and HLA-DR mRNA expressions were decreased in children with HFMDs on d1 of treatment (F=310.4, 81.5 and 545.4, P<0.01).After treatment, Th17 levels in mild group, severe group and surviving children of critically ill group were decreased, CD4 +CD25 +Treg and HLA-DR mRNA expressions were increased, while in fatal cases, Th17 level was still on the rise, and CD4 +CD25+Treg and HLA-DR mRNA expressions were still decreasing. After 10 d of treatment, the difference in Th17 and CD4 +CD25 +Treg levels among mild group, severe group, surviving children of critically ill group and control group was of no statistical significance ( P >0.05), but Th17 level in fatal was still higher and CD4 +CD25 +Treg level was still lower than those in control group (t=16.4 and 12.0, P<0.05).After 10 d of treatment, HLA-DR mRNA expressions in mild group and severe group were increased to the normal level.HLA-DR mRNA expression in surviving patients of critical ill group was still lower than that in mild group and severe group (P<0.05), but was higher than that in fatal patients (t=7.8, P<0.05).Pearson correlation analysis showed that, HLA-DR mRNA was negatively correlated with Th17 level (r=-0.770, P<0.01), and positively correlated with CD4 +CD25 +Treg level (r=0.883, P<0.01).Conclusion The expressions of Th17, CD4 +CD25 +Treg cells, and HLA-DR mRNA are correlated with the severity of HFMD, and may be used for evaluation of disease severity and prediction of disease outcomes.

Objective To evaluate the performance of transfection with a complex plasmid encoding green fluorescent protein tagged CatD (GFP-CatD)in researches on chronic photodamaged fibroblasts. Methods Human dermal fibroblasts (HSFs)were irradiated with ultraviolet A (UVA)at 25 J/cm2 once a day for 21 consecutive days to establish a chronic photodamaged cell model. A plasmid encoding GFP-CatD was constructed and transfected into some chronic photodamaged fibroblasts (experimental group). The photodamaged HSFs receiving no treatment served as the blank control group, and those transfected with the negative plasmid encoding GFP only as the negative control group. After additional culture, fluorescence microscopy and Western-blot analysis were performed to observe and measure the expression of GFP-CatD in HSFs respectively, flow cytometry and methyl thiazolyl tetrazolium (MTT)assay to evaluate the apoptosis and proliferation of chronic photodamaged fibroblasts respectively. Results Fluorescence microscopy showed the expression of GFP-CatD in cytoplasm of chronic photodamaged fibroblasts at 96 hours after transfection with the GFP-CatD-encoding plasmid. Western-blot analysis revealed that the expression of CatD in the experimental group was 1.28 times that in the blank control group. There were no significant differences in the apoptosis rate(4.29% ± 1.30%vs. 3.03% ± 1.70% , P > 0.05)or proliferative rate (45.20% ± 4.70% vs. 43.60 ± 3.90% , P > 0.05)between the experimental group and blank control group. Conclusion CatD could be traced in chronic photodamaged fibroblasts with no changes in biological activity or cell cycle after transfection with the GFP-CatD-encoding complex plasmid.

Objective To investigate the effect of ultraviolet A (UVA) radiation on the expression and secretion of cathepsin G (CatG) by human dermal fibroblasts.Methods Dermal fibroblasts were isolated from the foreskins of boys,and subjected to primary culture and subculture.After 10 or less passages,the fibroblasts were collected and divided into several groups to be irradiated with 10 J/cm2 UVA followed by 24,48 and 72 hours of additional culture,or be irradiated with 10,20 and 30 J/cm2 UVA followed by 24 hours of additional culture,with those receiving no treatment serving as the control group.Subsequently,cells and culture supernatant were collected,real time PCR and Western blot were performed to detect the expressions of CatG mRNA and protein respectively in these cells,and enzyme-linked immunosorbent assay (ELISA) was conducted to measure the expression of CatG protein in the culture supernatant of these cells.Results Compared with the control group,the fibroblasts irradiated with 10 J/cm2 UVA showed a significant increase at 24,48 and 72 hours in the expressions of CatG mRNA (0.376 ± 0.014 vs.0.183 ± 0.003,0.308 ± 0.022 vs.0.185 ± 0.005,0.296 ± 0.032 vs.0.182 ± 0.004,respectively,all P＜ 0.05) and protein (1.80 ± 0.12 vs.0.96 ± 0.06,1.41 ± 0.17 vs.0.95 ± 0.22,1.27 ± 0.09 vs.1.00 ± 0.14,respectively,all P ＜ 0.05),as well as in the supernatant level of CatG protein ((161.35 ± 7.55) vs.(122.45 ± 6.46) ng/L,(141.76 ± 2.95) vs.(124.17 ± 6.15) ng/L,(139.63 ± 3.04) vs.(121.72 ± 3.17) ng/L respectively,all P ＜0.05),with the strongest increase observed at 24 hours.At 24 hours after 10,20 and 30 J/cm2 of UVA radiation,the expression of CatG mRNA in irradiated fibroblasts was 1.90,2.51 and 3.04 times respectively (all P ＜ 0.05),the expression of CatG protein was 1.88,3.97 and 4.72 times respectively (P ＜ 0.05),and the supernatant level of CatG protein was 1.36,1.50 and 1.66 times respectively (P ＜ 0.05),that in the control group,and there was an increasing trend in all the above three parameters with increasing dose of UVA.Conclusion Acute UVA radiation can promote the expression and secretion of CatG by human dermal fibroblasts.

Objective To investigate the effects of repetitive ultraviolet A(UVA) radiation on the endocytosis and intracellular degradation of extracellular elastin by human dermal fibroblasts(HDFs), and to explore the mechanism responsible for elastin accumulation in photoaging skin. Methods Cultured HDFs were divided into two groups:repetitive UVA radiation group treated with UVA radiation(9.9 J/cm2)once a day for seven consecutive days to establish a chronic photodamage model, and blank control group receiving no treatment. For the verification of the model, flow cytometry was performed to detect cell cycle, and senescence-associated β-galactosidase staining to determine the degree of cell senescence. Fluorescent tracer technique and conjugated polymer-based fluorescence quenching technique were conducted to observe the endocytosis and intracellular degradation of extracellular elastin in culture media by HDFs in these groups. Results Compared with the blank control group, the repetitive UVA radiation group showed increased proportions of G1-phase cells and senescent cells, which confirmed the successful establishment of chronic photodamage model in the repetitive UVA radiation group. After coculture with elastin for 24, 48 and 72 hours, the endocytosis rate of elastin was 10.0% ± 1.4%, 27.8% ± 4.2% and 39.9% ± 4.1% respectively in the blank control group, 9.9% ± 1.6%, 28.3% ± 5.1% and 42.0% ± 5.7% respectively in the repetitive UVA radiation group, with no significant difference between the two groups at the three time points (all P > 0.05). The percentage of cells showing intracellular degradation of extracellular elastin was significantly lower in the repetitive UVA radiation group than in the blank control group (4.2% ± 1.1% vs. 7.7% ± 0.9% at 24 hours, 16.5% ± 2.4% vs. 22.8% ± 1.8% at 48 hours, 26.7% ± 2.6% vs. 33.9% ± 3.1% at 72 hours, all P < 0.05). Conclusions UVA radiation at 9.9 J/cm2 for 7 consecutive days can decrease the intracellular degradation of extracellular elastin by HDFs, but has no obvious effect on endocytosis of elastin.