Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.

Objective To discuss the clinical effects of treatment of femoral intertrochanteric fracture by DHS internal fixation. Methods 42 cases with femoral intertrochanteric fracture were treated by DHS internal fixation. According to AO classification, there were 16 cases of type A1,14 A2,and 12 A3. Results 36 patients were followed up for 6 to 18 months, with an average of 10 months. Their results were satisfactory. Conclusion Treatment of femoral intertrochanteric fractures with DHS internal fixation is an effective method.

Objective To explore the effects of targeted silencing of the chemokine receptor 7 (CXCR7)gene on the invasion and migration of the melanoma cell line M14. Methods Western-blot analysis was performed to determine the protein expression of CXCR7 in melanoma cell lines M14 and A375, and CXCR7-overexpressing M14 cells were used in this study. Cultured M14 cells were divided into three groups: experimental group transfected with a small interfering RNA(siRNA)targeting CXCR7(CXCR7-siRNA), negative control group transfected with a negative control siRNA, blank control group receiving no treatment. Real-time quantitative PCR and Western-blot analysis were conducted to determine the mRNA and protein expressions of CXCR7 respectively in M14 cells, Transwell chambers were used to evaluate the invasive activity of M14 cells, and wound healing assay to estimate the migratory activity of M14 cells. Results The experimental group showed significantly decreased mRNA and protein expressions of CXCR7 compared with the negative control group and blank control group (CXCR7 mRNA: 0.412 ± 0.023 vs. 1.211 ± 0.117 and 1.000 ± 0.102, F = 30.068, P = 0.001; CXCR7 protein: 0.144 ± 0.005 vs. 1 and 1.016 ± 0.004, F =11 485.5, P = 0.000). The number of M14 cells crossing the polycarbonate membrane per high-power field (× 200)was significantly smaller in the experimental group than in the negative control group and blank control group (20.617 ± 1.503 vs. 42.000 ± 6.018 and 43.627 ± 2.152, F = 32.416, P = 0.001). Similarly, the number of migrating M14 cells in wound healing assay was significantly decreased in the experimental group compared with the negative control group and blank control group (15.00 ± 1.10 vs. 44.90 ± 2.20 and 45.30 ± 2.30, F = 2 411.945, P = 0.000). Conclusion Targeted silencing of the CXCR7 gene can significantly inhibit the invasion and migration of M14 cells in vitro, which may provide a potential target for the treatment of cutaneous melanoma.

Objective To evaluate the role of the mitochondrial ATP-sensitive potassium (mito-KATP)channel in sevoflurane preconditioning-induced delayed cardioprotection against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Seventy-two adult male Sprague-Dawley rats were randomly divided into 6 groups (n =12 each):control group (group CON),I/R group,sevoflurane control group (group SEVO),sevoflurane preconditioning group (group SWO P),5-hydroxydeconoate (5-HD) + sevoflurane preconditioning group (group 5-HD+ SWOP) and 5-HD control group (group 5-HD).The rats were exposed to 33％ pure oxygen for 1 h in groups CON and I/R.The rats were exposed to 2.5％ sevoflurane for 1 h in groups SEVO and SWOP.5-HD (a mito-KATP channel inhibitor) 10 mg/kg was injected intraperitoneally 30 min before sevoflurane preconditioning in group 5-HD + SWOP.5-HD 10 mg/kg was injected intraperitoneally in group 5-HD.The hearts were immediately removed and perfused in a Langendorff apparatus.The hearts were made globally ischemic for 30 min followed by 120 min reperfusion in groups I/R,SWOP,5-HD + SWOP and 5-HD.The expression of phosphorylated protein kinase C-epsilon (p-PKC-ε) and caspase-8 was measured by Western blot immediately before ischemia (T0) and at 120 min of reperfusion (T1).The myocardial infarct volume was measured by TTC staining.Results Compared with group CON,the myocardial infarct volume was significantly increased at T1 in groups I/R,SWOP,5-HD +SWOP and 5-HD,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP and at T1 in groups I/R,SWOP,5-HD + SWOP and 5-HD,and caspase-8 expression was down-regulated at T1 in group SEVO (P ＜0.05).Compared with group I/R,the myocardial infarct volume was significantly decreased at T1 in groups SWOP and 5-HD + SWOP,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP,and caspase-8 expression was down-regulated at T1 in group SWOP (P ＜ 0.05).Compared with group SWOP,the myocardial infarct volume was significantly increased,p-PKC-ε expression was down-regulated at T0,and caspase-8 expression was up-regulated at T1 in group 5-HD + SWOP (P ＜ 0.05).Conclusion The mito-KATP channel is involved in sevoflurane preconditioning-induced delayed cardioprotection against I/R injury in isolated rat hearts through upregulation of p-PKC-ε expression before ischemia and inhibition of cell apoptosis during reperfusion.

Objective To analyze the clinic-pathological data and peritubular capillary (PTC) injuries of malignant nephrosclerosis (MN) patients and their correlations with the long term renal survival.Methods This was a retrospective cohort study of 52 MN patients in Peking Union Medical College Hospital from January 2003 to March 2012.Their clinical data and renal biopsy samples were carefully studied.CD34 staining was performed to evaluate the PTC area,using Benign nephrosclerosis (BN,n=17) patients and glomerular minimal lesions (GML,n=19) patients as controls.Multivariate Cox proportional hazard model was used to identify the potential independent risk factors for long term renal survival.Results Fifty-two MN patients were enrolled.The sex ratio of male to female was 12:1 and the average age was (34.0±8.2) years.The maximum blood pressure (SBP/DBP) was (230.4 ± 25.0)/(156.4 ± 20.6) mmHg,companied with significant loss of eGFR and proteinuria.Glomerular sclerosis index,tubular atrophy and interstitial fibrosis correlated with eGFR and proteinuria (P ＜ 0.05).After aggressive treatment,BP control rate improved significantly (76.9％ vs 3.7％,P ＜0.01),Scr [(376.4±263.8) μmol/L vs (486.8±375.7) μmol/L,Wilcoxon test,P＜ 0.01] and proteinuria [(1.10±0.70) g/24 h vs (2.04± 1.26) g/24 h,P ＜ 0.01,n=21] also improved.PTC area in MN patients was significantly lower than those in BN patients and GML patients,and it correlated well with Scr (r=-0.553,P=0.001) and eGFR (r=0.476,P=0.004).The median follow-up time was 74 months,the cumulative renal survival rate at 1 year,5 year and 10 year was 90％,64％ and 23％,respectively.Kaplan-Meier analysis showed that the patients with higher PTC area had longer renal survival time [(114.8± 12.4) months vs (63.0±8.3) months, x2=5.312,P ＜ 0.05].Univariate Cox proportional hazard model found that unsatisfied BP control,eGFR ＜ 30 ml · min-1 · (1.73 m2)-1 upon discharge,lower PTC area,severer tubular-interstitial damage and anemia were associated with poor renal outcome.Multivariate Cox model showed that unsatisfied BP control (RR=3.89,95％ CI 1.75-8.65,P=0.001),eGFR ＜ 30 ml · min-1 · (1.73 m2)-1 upon discharge (RR=4.27,95％ CI 1.40-13.09,P=0.011) were independent risk factors for long-term renal survival.Conclusions The correlation between PTC area and renal functions in MN patients are much better than that of classic vascular changes.Unsatisfied BP control and eGFR ＜ 30 ml · min-1 · (1.73 m2)-1 upon discharge are independent risk factors for long-term renal survival.

Objective:To explore the prolonged therapeutic regimen for patients with plaque psoriasis, who showed a positive response to 4-week treatment with tazarotene/betamethasone dipropionate cream, but were not completely cured.Methods:A multicenter, randomized, open-labelled, parallel-controlled clinical study was conducted. A total of 232 patients with plaque psoriasis were collected, who showed a positive response to previous 4-week treatment with 0.05%/0.05% tazarotene/betamethasone dipropionate cream, but were not completely cured with the psoriasis area and severity index[PASI] improvement rate being 50%-90%. At week 5, they were randomly and equally divided into 2 groups: test group receiving treatment with 0.05%/0.05% tazarotene/betamethasone dipropionate cream once a day, and control group receiving a sequential regimen of 0.05% tazarotene gel on weekdays once a day followed by 0.05%/0.05% tazarotene/betamethasone dipropionate cream on weekends once a day. After 2-and 4-week prolonged treatment, the efficacy and safety of the 2 therapeutic regimens were evaluated and compared. Measurement data were compared between 2 groups by using covariance analysis or t test, and enumeration data were compared by using chi-square test. Results:From the 5th to the 8th week, 200 out of the 232 patients completed the treatment. Data collected from 110 patients in the test group and 112 in the control group were enrolled into the full analysis set, and those from both 113 patients in the test group and control group were enrolled into safety analysis set. After consecutive 6-and 8-week treatment, the decline rates of the PASI score were 73.05% ± 16.69% and 78.46% ± 15.40% respectively in the test group, which were significantly higher than those in the control group (66.73% ± 21.77%, 67.02% ± 34.19%, respectively, both P < 0.05) . After 6-week treatment, the proportion of subjects who achieved PASI90 was significantly higher in the test group (14 cases, 12.7%) than in the control group (5 cases, 4.5%, χ2=4.842, P=0.028) ; After 8-week treatment, the proportions of subjects who achieved PASI75 and PASI90 (61.8%, 23.6%, respectively) were significantly higher in the test group than in the control group (48.2%, 12.5%, respectively, both P < 0.05) . During the consecutive 8-week treatment, there was no significant difference in the incidence rate of adverse reactions between the test group (15.0%) and control group (23.9%, χ2=2.822, P=0.093) . Conclusion:For patients who showed a positive response to 4-week treatment with 0.05%/0.05% tazarotene/betamethasone dipropionate cream, but were not completely cured, the continuous use of 0.05%/0.05% tazarotene/betamethasone dipropionate cream for 4 weeks is a superior therapeutic regimen compared with the sequential regimen of 0.05% tazarotene gel followed by 0.05%/0.05% tazarotene/betamethasone dipropionate cream.