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 inhibition effects of c erbB 2 and c raf 1 antisense oligodeoxynucleotides (ASODN) combined transfection on the human ovarian epithelial cancer transplanted subcutaneously in nude mice Methods There were 7 groups: normal control group,c erbB 2 sense observed group,c raf 1 sense observed group,c erbB 2 antisense observed group,c raf 1 antisense observed group,whole dose combined group,half dose combined group Human ovarian epithelial cancer cells SKOV3 were treated by different oligodeoxynucleotides,then transplanted subcutaneously in nude mice,respectively The changes of tumor volume were observed and the tumor growth inhibitory rate was calculated Results There was no difference between sense observed group and normal control group There was a larger growth inhibitory rate in whole dose combined group and half dose combined group,the first time that can be detected was 13 7 days and 15 2 days,and the maximum tumor growth inhibitory rates were 61 1% and 71 3%,respectively Conclusions The results suggested that ASODN combined transfection can inhibit the tumorigenesis of ovarian epithelial cancer cells in nude mice,it may be a more useful gene therapy for the ovarian epithelial carcinoma

Objective To explore the effect of down-regulating epidermal growth factor receptor (EGFR) expression by RNA interference on the proliferation of ovarian carcinoma SKOV3 cells. Methods The recombinant eukaryotic expression plasmids pGenSil-HK,pGenSil-EGFR1 and pGenSil-EGFR2 were constructed and transfected into SKOV3 cells respectively. The mRNA and protein expressions of EGFR in SKOV3 cells were detected by RT-PCR and Western blotting,respectively. The cell cycle and apoptosis were evaluated by flow cytometry. The proliferation of SKOV3 cells was determined by clone formation assay and MTT assay. Results We successfully constructed the recombinant eukaryotic expression plasmids pGenSil-HK,pGenSil-EGFR1 and pGenSil-EGFR2 and transfected into SKOV3 cells. Three cell clones were screened by G418. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK,the expressions of EGFR in SKOV3 cells transfected with pGenSil-EGFR1,pGenSil-EGFR2 were inhibited significantly at both mRNA and protein levels,with an inhibitory rate of 41.87% and 68.07% for EGFR mRNA and of 45.21% and 70.25% for EGFR protein respectively. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK,the cell apoptotic rate was significantly increased significantly,the cell cycle was arrested in G1 phase and S phase decreased significantly in pGenSil-EGFR2 SKOV3 cells (P

Objective To observe the effect and side effects of concurrent chemoradiotherapy for stage ⅡB～ⅢB cervical cancer. Methods 126 patients with stage ⅡB～ⅢB cervical cancer were randomly allocated into 2 groups. Concurrent chemoradiotherapy group: radiotherapy carried out same as the neoadjuvant chemotherapy group, i.e.firstly with cisplatin 20 mg iv d1-5, 5-Fu 750 mg iv d1-5, repeated every 28 days, total 4 cycle; the neoadjuvant chemotherapy group i.e.firstly with cisplatin 20 mg iv d1-5, 5-Fu 750 mg iv d1-5, repeated every 28 days, total 2 cycle, after chemotherapy received routine radiotherapy 2 Gy per day, 5 times a week to a total dose of 30 Gy with 192Ir brachytherapy, 7 Gy per week. When total dose reached 46 Gy, the middle field was shielded by plumbum, then continuous radiotherapy, total dose reach A point 65-70 Gy, B point 50-56 Gy. Results All patients were followed-up for more than five years. The follow-up rate was 94.4 %. In concurrent chemoradiotherapy group the 3 year survival rate and the 5 year survival rate were 82.8 %, 65.6 %, In neoadjuvant chemotherapy group the 3 year survival rate and the 5 year survival rate were 67.7 %, 46.8 %. There was a significant difference in two groups (X2=3.86, P＜0.05; X2=5.01, P＜0.05), no significant difference in toxicity-side effect. Conclusion Concurrent chemoradiotherapy for advanced cervical cancer can significantly improve the 3-year and 5-year survival rate and has little increase in toxicity-side effect.

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 observe the NTBC dependence of Fah-knockout mice and study the biological characteristics in order to use the model more effectively.Methods Examine the progressive changes in body weight, survival time, liver pathology and serological markers after the NTBC withdrawal.Results After removing of NTBC, Fah-knockout mice lost their body weight gradually, and finally died in 5 to 7 weeks, along with increased serum ALT, AST levels and deformation of the hepatocytes.Conclusions Fah-knockout mice have a strong drug dependence of NTBC and could be the ideal model to hereditary tyrosinemia type I and other liver injury.

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 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 clinical significance of apoptosis of B cells in the peripheral blood of patients with rheumatoid arthritis (RA).Methods The proportions of B cells in the peripheral blood (PB) of 51 active and 30 remission patients with RA and 80 healthy controls were detected by flow cytometry.B cells in the PB of 10 active,10 remission and 10 healthy individuals were isolated by MACS.The apoptosis of cultured B cells,which were collected at 24,48,72,96 h respectively,were assessed by flow cytometry.ANOVA,t test and,Spearman correlation analysis were used for statistical analysis.Results The proportions of B cells marked as CD19 and CD22 in the PB of active and remission patients with RA and healthy controls were (26±11)％,(12±8)％,(10±7)％,(26±10)％,(12±8)％,(11±5)％ respectively.The proportions of B cells in the PB of active patients was significantly higher than that of remission patients and healthy controls (P＜0.01).There was positive correlation between B cell proportion in the PB of active patients and DAS 28 and IgG level.The proportion of apoptosis of B cells in the PB with active patients was less than healthy controls.Conclusion The pathway of apoptosis of B cells in the PB of active patients is inhibited,which could increase B cell proportion.Moreover,the high proportion of B cells in the PB of active patients is closely related to disease activity.

Objective To evaluate clinical efficacy of treating femoral intertrochanteric and subtrochanteric fractures combined with femoral shaft fractures using anatomic locking plate.Methods From January 2009 to June 2011,we treated 72 cases of femoral intertrochanteric and subtrochanteric fractures combined with femoral shaft fractures using anatomic locking plate.Sixty-four cases were followed up.There were 50 males and 14 female,with an average age of 42.8 years (range,21-79).All patients suffered from closed femoral unilateral fractures.The interval between injury and surgery was 2 h-16 d (average,4.6 d).All patients were followed up at regular interval.During the follow-up period,clinical and radiographic data were recorded.The clinical efficacy was evaluated with Harris hip function score.Results The mean operative time was 65 min (range,45-120 min); the mean blood loss was 210 ml (range,50-650 ml).All patients began to walk with crutches 3-35 d after surgery.During the follow-up period,no infection,deep vein thrombosis,screwed cut-out and implant failure occurred in all patients.Coxa vara with shortening deformity was noted in 2 cases,solid bone union was found in all the cases.The mean time of fracture healing was 5.2 months (range,3.6-10.5 months).According to Harris hip score,45 cases were classified as excellent,14 as good and 5 as fair,with excellent and good rate being 92.19％ (59/64).Conclusion Anatomic locking plate fixation provides stable fixation,with a high union rate and a minimal complication rate in treatment of intertrochanteric and subtrochanteric fractures combined with femoral shaft fracture.