Objective To investigate the effects of botulinum toxin type A on the proliferation,apoptosis and ERK/MAPK signaling pathway of human hypertrophic scar fibroblasts in vitro.Methods Fibroblasts were isolated from human hypertrophic scar and cultured by tissue explant method.The cells were divided into control and botulinum toxin type A groups.The botulinum toxin group A was cultured in DMEM medium containing 0.4U/L botulinum toxin type A.The control group was cultured with DMEM alone.Cell proliferation was measured by CCK8 kit at days 1,3,5 and 7 and compared within groups.Apoptosis of fibroblasts was detected by flow cytometry which treated with botulinum toxin and control group.Western blot was used to detect the relative expression of collagen Ⅰ,p-ERK1/2 and total ERK protein in fibroblasts of control,botulinum toxin type A and U0126 groups.Results Botulinum toxin type A could significantly inhibit the proliferation of fibroblasts,the number of cells was only 68.9％ in the control group at 7th days.The apoptotic rate of botulinum toxin type A was 35.9％,which was significantly higher than that of the control group.ERK 1/2 protein expression in the three groups was not significantly different (P ＞ 0.05),while p-ERK 1/2 expression in the control group was still significantly higher,but the p-EKR 1/2 in botulinum toxin type A and U0126 group was significantly inhibited.Collagen Ⅰ was highly expressed in the control group,which was significantly higher than that in the botulinum toxin type A and U0126 groups (P ＜ 0.05).Conclusion Botulinum toxin type A can inhibit the ERK/MAPK signaling pathway and increased fibroblasts apoptosis,decreased proliferation activity and collagen Ⅰ secretion.

Objective To study the variation and prognostic value of serum heparin-binding protein (HBP),a potent inducer of vascular leakage in septic shock.Methods A total of 82 sepsis patients admitted to central ICU from August 2011 to July 2012 were enrolled in the prospective study.Eighty-two sepsis patients were divided into septic shock group (group A,n =39) and sepsis without shock group (group B,n =43).At the same time,another 30 shock patients without sepsis were enrolled as group C and 35 patients without sepsis and shock were enrolled as group D.Blood samples obtained within 24 hours after enrollment,and the concentrations of HBP were measured by ELISA and lactate by electrode method.Comparisons of HBP,lactate and APACHE Ⅱ score between groups were carried out by Studers't test,and the early prognostic value in septic shock and mortality was studied by receiver operating characteristic (ROC) curve.Results Patients in group A (t =3.862,P＜0.001) and group B(t =5.193,P＜ 0.001) both had higher HBP level than patients in group D.HBP level in group A patients was also higher than that of group B (t =3.270,P =0.002).Septic shock patients (group A) had higher HBP than patients of group C (t =3.029,P =0.004).The area under curve (AUC) of HBP predicting septic shock patients was 0.834,with sensitivity 0.795,specificity 0.795.The AUC of HBP predicting 28-day mortality of all enrolled patients was was 0.680,and the AUC of predicting 28-day mortality of sepsis patients was 0.784.Conclusions HBP levels were increased in septic patients,especially in septic shock patients.HBP is a better predictor than traditional predictors like lactate and APACHE Ⅱ score for predicting septic shock.HBP is a good and specific evaluation marker for predicting septic shock and the mortality of sepsis patients.The measurement of HBP during the early stage of disease presents valuable information about diagnosis and treatment.

Objective To investigate the expression of serum sHLA-G in systemic lupus erythematosus (SLE) patients and the association with the disease activity.Methods The serum concentration of sHLA-G in SLE patients and healthy controls was measured with enzyme-linked immunosorbent assay.Results Significant higher sHLA-G levels were detected in patients of SLE than control group (P＜0.01),The serum concentrations of sHLA-G in active SLE patients were markedly higher than stable SLE patients (P＜0.01).The expression level of sHLA-G showed positive correlations with SLE activity index (SLEDAI)(r=0.30,P=0.01).There was no correlation between sHLA-G levels and serum concentration of Anti-dsDNA,C3,C4 and Anti-ANA in SLE patients (P＞0.05).Conclusion The level of serum sHLA-G is significantly increased in SLE patients.Positive correlations are observed between sHLA-G levels and SLEDAI.These data indicated that sHLA-G may play certain roles in the pathogenesis and progress in SLE.

Objective To investigate the expression of helper T cells (Th)17/regulatory T cells (Treg) balance associated factors in rheumatoid arthritis (RA) patients and their correlation with serum midkine (MK).Methods A total of 60 RA patients were divided into active RA patients (n=32) and inactive RA patients group (n=28).MK level in sera was detected by enzyme linked immunosorbent assay (ELISA) in 60 patients with RA and 30 healthy controls (HCs).The fraction of CD4+CD25+FOXP3+ Treg cells and IL-17+CD4+ Th17 cells in RA patients and healthy controls were determined by flow cytometry (FCM), and the expreasion of Foxp3, RORγt, Signal transducer and activator of transcription (STAT) 3 and STAT5 mRNA were detected with real-time polymerase chain reaction (PCR).Results were evaluated using ANOVA followed by q tests for comparisons of Th17 population between active RA patients, inactive RA patients and HCs, t test was used for comparing of Foxp3, RORγt, STAT3, STAT5 mRNA between RA group and HCs.The correlations between serum MK concentration and peripheral Treg cells, Th17 cells, Foxp3, RORγt, STAT3, STAT5 mRNA were analyzed by Pearson's correlation analysis.Results The percentages of Treg cells from active RA patients, inactive RA patients and HCs were significantly different (F=129.6, P＜0.01), the percentages of Treg cells of active RA patients [(1.41±1.05)％] were lower than that of the inactive RA patients [(3.6±1.6)％;q =7.92, P＜0.05] and healthy group [(7.7±1.7)％;q=22.45, P＜0.05], and there was significant difference between healthy group and inactive RA group (q=14.53, P＜0.05).The percentages of Th17 cells of the three groups were also significantly different (F=36.3, P＜0.01),the percentage of Th17 cells of active RA patients [(1.84±1.01)％] was significantly higher than that of inactive RA patients [(0.71±0.28)％;q=9.59, P＜0.05] and healthy group (0.53±0.16)％ [(P＜0.05;q=1 1.10, P＜0.05], there was no significant difference between the inactive RA group and healthy group (q=1.51, P＞0.05).The expression of RORγt and STAT3 mRNA in RA patients was higher than that of healthy controls (t=5.84, P＜0.01;t=4.52, P＜0.01).The expression of Foxp3 and STAT5 mRNA in RA patients were lower than healthy controls (t=6.01, P＜0.01;t=2.18, P＜0.05).Serum MK values were correlated with STAT5 (r=-0.55, P＜0.01), but not with Foxp3, RORγt, STAT3 mRNA or the percentage of Treg/Th17 cells.Conclusion Serum MK expression and the percentage of Th17 cells increase, while the percentage of Treg cells decrease in RA patients.Serum MK values are negatively correlated with STAT5 mRNA which is associated with Th17/Treg balance.This may be important in the pathogenesis of RA.

Objective To investigate the relation between the apotosis of B cells in the peripheral blood (PB) and the expression of interleukin (IL)-17 in patients with rheumatoid arthritis (RA).Methods The proportions of apoptosis of B cells in the PB of 80 patients with RA and 80 healthy controls were measured by flow cytometry.B cells in the PB of 20 RA and 20 healthy individuals were isolated by MACS and Western blotting was used to detect the Bcl-2 and Caspase-3 protein levels.IL-17 levels were detected by enzyme-linked immunosorbent assay (ELISA).T-test and linear regression were used to analyze the data.Results The proportions of apoptosis of B cells in the PB of patients with RA and healthy controls were (14±6)％ and (24±9)％ respectively.The rate of apoptosis of B cells in patients with RA was significantly less than healthy controls (t=2.737,P=0.021).The Bcl-2 protein level of B cells in the PB of patients with RA group was significantly higher than that of control group (26±10,12±6,P＜0.01).Conversely,the Caspase-3 protein level of B cells in the PB of patients with RA group was significantly lower than that of the control group (16±7,31±12,P＜0.01).ELISA detected elevated level of serum IL-17 in the patients with RA as compared with controls [(69±19),(27±10) pg/ml,t=4.631,P=0.014].There was a negative correlation between the level of IL-17 and apoptosis of B cells in patients with RA (r=0.36,P＜0.01).Conclusion The elevated bcl-2 and reduced caspase-3 of B cells in patients with RA further proves there is abnormal apoptosis of B cells in RA patients.There is negative correlation between the expression of IL-17 and apoptosis of B cells in patients with RA and IL-17 can inhibit B cell apoptosis.

Objective To study and compare the effects of Fentanyl transdermal pain patches and Pethidine in postoperative analgesia of malignant tumor and its nursing methods.Methods 100 patients with malignant tumor in our hospital from January 2008 to November 2010 were selected as research object,all the patients were randomly divided into group A (Fentanyl transdermal pain patches group) and group B (Pethidine group)with 50 cases in each group,then the VAS score 2,4,8,24 and 48h after the surgery,incidence of adverse reactions,satisfaction rate and serum catecholamine,cortisol,blood glucose before and after the surgery were analyzed and compared,and all the patients were given corresponding nursing measures.Results The VAS score of group A at 2,4,8,24 and 48h after the surgery were all better than those of group B,incidence of adverse reactions was lower than that of group B,satisfaction rate was higher than that of group B,serum catecholamine,cortisol,blood glucose after the surgery were all lower than those of group B,there were significant differences.Conclusions The effects of Fentanyl transdermal pain patches in postoperative analgesia of malignant tumor is stable,and it gets good reputation from the patients,the effects is obvious combined with nursing.

Objective To investigate the diagnostic values of serum free fatty acids (FFA) and urine podocalyxin (PCX) in the patients with type 2 diabetic nephropathy(DN).Methods A total of 120 patients with type 2 diabetes mellitus (DM) were enrolled,and they were divided into three groups,normal albumin group(NA),microalbuminuria group(MA) and heavy albuminuria group(HA),based on the urinary albumin-creatinine ratio(UACR).In addition,40 healthy persons participated in medical examination were selected as the control group.Serum FFA levels were detected by a biochemical analyzer and urine PCX levels by ELISA.The correlation between them was analyzed by Pearson correlation,and their diagnostic values in type 2 DN were evaluated by the receiver operating characteristic curve (ROC).Results Serum FFA levels in the NA,MA,HA and control groups were (0.61 ± 0.14),(0.81 ± 0.13),(0.95 ± 0.18) and (0.49 ± 0.11) mmol/L,respectively,and urine PCX levels were (1.86 ± 0.45),(4.47 ± 1.48),(6.72 ± 1.40)and(1.38 ±0.24) ng/mL,respectively.The levels of serum FFA and urine PCX in type 2 DM patients were significantly higher than those in the control group(P ＜ 0.05),and increased with the progression of type 2 DM.Pearson correlation analysis showed that serum FFA levels were positively correlated with urine PCX levels(r =0.73,P ＜ 0.05).The sensitivity of serum FFA,urine PCX and combined detection in the diagnosis of type 2 DN were 74.1％,80.6％ and 89.5％,respectively,and the latter significantly higher than the former two (P ＜ 0.05).Conclusion Serum FFA and urine PCX levels increase significantly in diabetic patients with renal injury,and both of them have important clinical values in the diagnosis of type 2 DN.

Objective To explore the effect and action principle of extracorpoporeal shock wave(ESW) combine hyperbaric oxygen(HBO) in bone nonunion treatment .Methods Totally 50 standard New Zealand white rabbits were chosen ,and 48 rabbits were successfully made to models .Then ,they were divided into four groups by using the random number table ,12 in each group . The group A was in ESW combine HBO group ;The group B was in hyperbaric oxygen group ;The group C was in pure ESW group ;The group D was in control group .X‐ray inspected before treatment and after treatment in 4 、8 、12 weeks .The calcium con‐tent was checked ,the osteoblast in bone callus was observed by the optical microscope ,and the data was analyzed by statistics .Re‐sults There was a significant difference in 4 ,8 ,12 weeks between group A and group B ,C ,D in the nonunion gap(P< 0 .05) ,there was significant difference in 4 ,8 ,12 weeks between group A and group B ,C ,D in the generation bone callus(P< 0 .05) .Callus calci‐um content of group A was higher than group C ,the difference was statistical significance(P< 0 .05) ,there was significant differ‐ence in 4 ,8 weeks between group A and group C in callus osteoblast count(P < 0 .05) .Conclusion ESW combine HBO treatment for bone nonunion is better than pure ESW therapy ,simple hyperbaric therapy has no obvious help for the treatment of bone nonun‐ion ,HBO can be used as a good synergy method in the extracorporeal shock wave treatment of bone nonunion .

Objective To establish a method for detecting urinary vanillylmandelic acid (VMA), homovanillic acid (HVA) and creatinine (Cr) simultaneously by high performance capillary electrophoresis (HPCE). Methods The separations were carried out using a 120 mmol/L phosphate buffer (pH 6.80) in a fused-silica capillary tube of 47 cm×75 μm I.D. by capillary zone electrophoresis (CZE). Injections were made by using the pressure mode for 4 s at 1 p. s. i. after samples were centrifuged and diluted. The detections were monitored by a diode-array detector (DAD) at 200 nm after samples were separated at a voltage of 20 kV. The method developed was validated systematically and applied to urine samples from healthy adults (n = 100) and children (n = 100) for establishing the reference ranges of VMA/Cr and HVA/Cr, respectively. Results Under these conditions, the separations of VMA, HVA and Cr could be completed within 13 min. The linearity ranges of VMA, HVA and Cr were 0-500, 0-500 and 0-4 000 μmol/L, respectively, with the correlation coefficients (r) between 0.997 2 and 0. 999 1 (P < 0.01). The detection limits (S/N= 3) were 1.0 μmol/L for VMA, 1.0 μmol/L for HVA and 50.0 μmol/L for Cr. The mean within-run (n = 10) CVs of migration time for VMA, HVA and Cr in urine were 0.58%, 0.56% and 0.25% respectively, while the mean between-run (n = 10) CVs of migration time were 0.95%, 1.00% and 0.48% respectively. The mean within-run (n = 10) CVs of peak area for VMA, HVA and Cr were 3.78%, 3.97% and 2.76% respectively, while the mean between-rim (n = 10) CVs of peak area were 4.60%, 4.08% and 4.42% respectively. The average recoveries were 98.36% for VMA, 93.56% for HVA and 98.85% for Cr. Other compounds in human urine such as catecholamines, 5-hydroxytryptamine and albumen didn't interfere with the assay. The correlation between CE method and HPLC method was good. And the correlation coefficients (r) of VMA and HVA were 0.954 9(P <0.01) and 0.945 1 (P < 0.01), respectively. Skewness distributions were presented for VMA/Cr and HVA/Cr in random urine from both adults and children, and the 95% reference ranges were established by the percentile method. For adults, the reference ranges of VMA/Cr and HVA/Cr were 0-4. 26 and 0-1.69 (μmol/mmol), respectively. For children, the reference ranges of VMA/Cr and HVA/Cr were 0-10.39 and 0-4.31 (μmol/mmol), respectively. Conclusions The CE method devised here for direct measurement of urinary VMA, HVA and Cr is simple, fast,precise and automatic with good repeatability. It is an ideal method for routine detection and mass screening of pheochromocytoma and neuroblastoms.

Objective To study the role of heparin-binding protein (HBP) in sepsis-associated acute respiratory distress syndrome (ARDS),and to evaluate the prognostic value of HBP in ARDS.Method Sixty seven sepsis patients were enrolled in the prospective study.According to whether present ARDS,patients were divided into two groups:ARDS group and non-ARDS group.Blood samples were obtained within 2 hours after patients were diagnosed with sepsis.We measured the level of interleukin-6,interleukin8 and HBP by ELISA,counted the number of polymorphonuclear neutrophils (PMN),and calculated Acute Physiology and Chronic Health Evaluation Ⅱ score.We compared interleukin-6,interleukin-8,PMN and HBP between two groups by Student's t test.Correlation analysis was studied between HBP and other indicators by Spearman rank correlation coefficient.We also studied the early prognostic value on sepsisassociated ARDS by establishing receiver operating characteristic (ROC) curve.Results Interleukin-6 (t=4.25,P＜0.01),interleukin-8 (t=10.10,P＜0.01),PMN (t=3.47,P=0.0009) and HBP (t =0.0225,P =2.336,all showed significant difference between ARDS group and non-ARDS group.Interleukin-6 (r=0.535,P=0.002),interleukin-8 (r=0.419,P=0.017) and PMN (r=0.419,P =0.017) all had positive correlation with HBP.The area under curve (AUC) of HBP predicting ARDS was 0.796.Conclusions HBP level elevated in sepsis-associated ARDS patients and were correlated with interleuki-6,interleuki-8 and PMN.HBP show a high value on predicting emergence of sepsis-associated ARDS.HBP may play an important role in sepsis-associated ARDS pathological process.From this study we can conclude one of possible ARDS pathogenesis:inflammatory mediators,such as interleuki-6,interleuki8,activate PMN.Subseqoently PMN releases HBP.Then HBP resolts in vascolar leakage,which is one of the basic ARDS pathology.