Objective To identify the role of microRNA-17(miR-17)in human glioma U87 cells invasion which may regulate expression of matriv metalloproteinase(MMP)-2.Methods U87 cells were cultured in vitro,while changes in cellular morphology were observed by phase contrast microscope.The miR-17 which might regulate the expression of MMP-2 was predicted by bioinformatics and identified using dual luciferase report system.Expressions of miR-17 and MMP-2 were determined using real-time PCR and Western blot after transfection of miR-17 mimics.The invasion of U87 cells was detected in vitro by Transwell chamber.Results Expression of MMP-2 was positive by immunofluorescence cytochemistry. Using dual luciferase reporter system,miR-17 could inhibit the expression of MMP-2 by binding to its mRNA 3′UTR. Results of real-time PCR and Western blot showed that over-expression of miR-17 down-regulated expression of MMP-2. The invasion of U87 cells was suppressed by over-expression of miR-17.Conclusion MiR-17 may negatively regulate expression of MMP-2 in human glioma U87 cells and inhibit cell invasion.

Objective To evaluate the role of TWIK-related K+ channel 1 (TREK-1) in reduction of cerebral ischemia-reperfusion (I/R) injury by sevoflurane preconditioning in mice.Methods Sixty male Kunming mice,aged 8 weeks,weighing 21-29 g,were randomly divided into 5 groups (n=12 each) using a random number table:sham operation group (group S),group I/R,sevoflurane preconditioning group (group SP),TREK-1 small hairpin RNA (shRNA) lentivirus + sevoflurane preconditioning group (group TSP),and negative control shRNA lentivirus + sevoflurane preconditioning group (group NSP).In S,I/R and SP groups,normal saline 15 μl was injected into the lateral cerebral ventricle at 1 μl/min.In TSP and NSP groups,TREK-1 shRNA lentivirus and negative control shRNA lentivirus 15 μl were injected into the lateral cerebral ventricle,respectively,at a rate of 1 μl/min.And 14 days later,S and I/R groups inhaled 100％ oxygen for 60 min,SP,TSP and NSP groups inhaled 2.4％ sevoflurane for 60 min,followed by 15 min washout by inhaling 100％ oxygen,and then cerebral I/R was produced by occlusion of right internal carotid artery for 2 h followed by reperfusion.At 24 h of reperfusion,neurological deficit was scored (NDS).The mice were then sacrificed,and brains were removed to determine the cerebral infarct size (IS),expression of hippocampal caspase-3 and cell apoptosis in brain tissues.Apoptosis index (AI) was calculated.Results Compared with group S,the NDS,cerebral IS,expression of hippocampal caspase-3 and AI were significantly increased in I/R,SP,TSP and NSP groups.Compared with group I/R,the NDS,cerebral IS and AI were significantly decreased,and the expression of hippocampal caspase-3 was down-regulated in SP,TSP and NSP groups.Compared with group SP,the NDS,cerebral IS and AI were significantly increased,and the expression of hippocampal caspase-3 was up-regulated in group TSP,and no significant change was found in the parameters mentioned above in NSP group.Conclusion Sevoflurane preconditioning reduces cerebral I/R injury through activating TREK-1 and inhibiting apoptosis in hippocampal neurons of mice.

Objective To establish a real‐time fluorescent polymerase chain reaction and detect 16S rRNA gene of Legionella strains isolated from sputum specimens of patients with pulmonary infection by using this method .Methods 16s rRNA gene of Le‐gionella was used to design primers and probes .The reaction system and reaction conditions were optimized and the specificity ,sen‐sitivity and repeatability of this method were verified by detecting Legionella pneumophila ,non‐Legionella pneumophila and other bacteria .A total of 557 sputum specimens of patients with pulmonary infection were detected ,and PCR‐digestion identification method was carried out as control .Otherwise ,sequences of 16S rRNA were verified in patients with positive detection results .Re‐sults The results showed that all reference strains of Legionella were positive ,while all of other bacteria were negative ,and the sensitivity was 102 CFU/mL .Among sputum specimens collected from 577 cases of patients with pulmonary infection ,the positive rate of Legionella detected by using real‐time fluorescent PCR and PCR‐digestion identification method was 23 .1% and 19 .9% re‐spectively ,while the positive rate was 17 .2% by verifying the sequences of 16s rRNA .There were no statistically significant differ‐ences of positive rate among the three methods(P>0 .05) .Conclusion The real‐time fluorescent PCR is fast and convenient in de‐tection of Legionella strains isolated from sputum specimens of patients ,which could be an assisted method for clinically diagnosing Legionella infection .

Objective To explore the expression of costimulatory molecules of CD28 and CD152 for patients with chronic hepatitis B(CHB).Methods Flow cytometric analysis was applied to detect the expression level of CD28/CD152 on CD4+ or CD8+ T lymphocyte subpopulations.Results Compared with control group,the percentage of CD4+CD28+ and CD8+CD28+ increased significantly(P

Objective To evaluate the clinical outcome in the treatment of humerus shaft comminuted fractures using limited open reduction and internal fixation combined with an external fixator.Methods Data of 80 patients with comminuted humerus shaft fractures treated from January 2005 to January 2013 were analysed retrospectively.All the patients underwent limited open reduction and internal fixation combined with an external fixator (treatment group) and open reduction and plate fixation (control group) according to the random number table.In the treatment group,there were 40 patients (28 males,12 females),at mean age of 33.5 years (range,21-54 years),with causes of injury including traffic accidents in five patients,falls in nine,crashes in seven and others in six.There were seven patients with open fractures and 33 with closed fractures.In the control group,there were 40 patients (25 males,15 females),at mean age of 32.9 years (range,19-55 years),with causes of injury including traffic accidents in 16 patients,tumbling in seven,crush in seven and others in ten.There were eight patients with open fractures and 32 with closed fractures.The operation time,intraoperative blood loss,bone union time and complications in both groups were recorded.Clinical efficacy was evaluated using the Stewart and Hundley standard.Results Mean follow-up was 19 months (range,15-24 months).Treatment and control groups showed significant differences in operation time [(55.5 ± 10.3) minutes vs.(120.5 ± 15.3) minutes],intraoperative blood loss [(120.4 ± 20.7) ml vs.(245.4 ± 26.7) ml] and bone union time [(11.6 ± 1.3) weeks vs.(14.9 ± 2.3) weeks] (P ＜ 0.05).Rate of incision infection was 8％ (3/40) in treatment group and 10％ (4/40) in control group (P ＞ 0.05).In treatment group the results were excellent in 31 patients and good in nine.In control group the results were excellent in 27 patients,good in nine,fair in one and poor in three.One patient with radial nerve injury after a second surgery for implant removal and two patients with osteomyelitis or bone nonunion were noted in control group.Conclusion Limited open reduction and internal fixation in combination with an external fixator is associated with small trauma,easy operation,short operation time,few bleeding,rigid fixation,early functional exercises and reduced bone nonunion for treatment of comminuted humerus shaft fractures,which exhibits great clinical value.

Objective To explore the effects of removing-stasis and resuscitating methods on patients with severe cerebral injury. Methods Retrospective analysis was performed with clinical data of 47 patients. All 47 patients were recruited into a control group(n=21) and a treatment group(n=26). The control group was treated with conventional western medicines; while the treatment group was additional treated with traditional Chinese medicine on the basis of the control group. Results The rate for resuscitation after one week's treatment was 73.08% and 57.14% in the treatment group and the control group respectively, showing significant differences (P<0.01). The total effective rate was 76.92% and 61.90% in the treatment group and the control group respectively, also showing significant difference (P<0.05) . Conclusion The method of removing-stasis and resuscitating can promote severe cerebral injury patients regain their consciousness, and improve their prognosis.

Objective To investigate the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) protein in the spinal cord neurons in diabetic neuropathic pain (DNP) in rats.Methods Male Sprague-Dawley rats,aged 2 months,weighing 180-200 g,were studied.Diabetes mellitus was induced by single intraperitoneal injection of streptozotocin (STZ) 60 mg/kg.Sixteen rats with DNP were randomly divided into 2 groups (n =8 each) using a random number table:DNP group and DNP+PTEN inhibitor bpv (pic) group (DPN-bpv group).Another 16 rats were equally and randomly divided into either control group (group C) or bpv group.In DNP-bpv and bpv groups,bpv (pic) 0.2mg/kg was injected intraperitoneally once a day within 14-28 days after injection of STZ.Before STZ injection (T1),and at 2,7,14,21 and 28 days after STZ injection (T2-6),the mechanical paw withdrawal threshold (MWT) was measured.After measurement of MWT,the rats were sacrificed,and the lumbar segments of the spinal cord (L4.5) were removed for determination of PTEN protein activity (by ELISA) and Akt (s473) phosphorylation (by Western blot).Results Compared with group C,the MWT was significantly decreased at T4-6,and the PTEN protein activity and Akt (s473) phosphorylation were significantly increased in DNP and DNP-bpv groups (P＜0.05 or 0.01).Compared with group DNP,the MWT was significantly increased at T6,and the PTEN protein activity and Akt (s473) phosphorylation were significantly decreased in group DNP-bpv (P＜0.05).Conclusion PTEN protein in the spinal cord neurons is involved in the maintenance of DNP in rats.

Objective To observe the effect of adenosine monophosphate activated protein kinase (AMPK) on attenuating inflammation in fibrosis induced hy acute ischemia reperfusion injury (IRI) in mice.Methods Forty eight male C57BL/6 mice were randomly divided into four groups:sham operation group (sham group),IRI group,AMPK inhibitor+IRI group (AMPK/IRI group) and normal saline+IRI group (NS/IRI group),12 mice each group.The mice with renal IRI were occluded for 30 min through clipping bilateral renal pedicle,then released renal perfusion.Mice in sham group were performed the separation of renal pedicle without clipping.Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI.At the 2 d after operation,6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr.The renal histopathological changes were observed through HE staining.The mRNA expression of IL-1β,IL-6 and TNF-α was detected by real time PCR,and the level of AMPK phosphorylation was detected by Western blotting.At the 14 d after operation,Collagen 1 (COL1),α-SMA and fibronectin (FN) were detected by immunofluorescence and Western blotting in 6 remained mice from each group.The degree of kidney fibrosis was observed through sirus red staining.Results Compared with those in sham group,tubular interstitial damage was aggravated (P ＜ 0.05),BUN and Scr were increased (P ＜ 0.05),the mRNA expression of IL-1β,IL-6 and TNF-α was increased at the 2 d after operation (all P ＜ 0.05),and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group (all P ＜ 0.05);the degree of kidney fibrosis and the expression of COL1,α-SMA and FN were increased obviously at the 14 d (all P ＜ 0.05).Compared with those in IRI group,in AMPK/IRI group tubular interstitial damage was aggravated (P ＜ 0.05),BUN and Scr were increased (all P ＜ 0.05),the mRNA expression of IL-1β,IL-6 and TNF-α was increased at the 2 d (all P ＜ 0.05),and the level of AMPK phosphorylation was decreased (P ＜ 0.05).Moreover,the degree of kidney fibrosis and the expression of COLI,α-SMA and FN were increased obviously at the 14 d in AMPK/IRI group (all P ＜0.05).Conclusions AMPK can ameliorate the acute renal ischemia reperfusion injury induce fibrosis in mice,and the mechanism may be related to the decrease of inflammatory reaction.

Objective To evaluate the effects of intrathecal TRESK gene recombinant adenovirus on inflammatory responses mediated by chemokine in the spinal cord of rats with neuropathic pain ( NP ) . Methods Thirty?six male Sprague?Dawley rats, weighing 200-250 g, were randomly divided into 6 groups (n=6 each) using a random number table: control group (group C); sham operation group (group S);NP group; TRESK?overexpressed adenovirus group ( group TRESK ); negative adenovirus group ( group Virus); normal saline group ( group NS) . Spinal nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats. In TRESK, Virus and NS groups, pAd∕CMV∕V5?DEST?TRESK 25 μl (109IU∕ml), negative adenovirus 25 μl and normal saline 25 μl were intrathecally injected, respectively. At 1 day before operation ( base?line, T0 ) and 1, 3, 7 and 14 days after operation ( T1-4 ) , the mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal latency were measured. Six rats in each group were sacrificed after measurement of pain threshold at T3 . The L4,5 segments of the spinal cords were removed for determination of monocyte chemotactic protein?1 ( MCP?1) , MIP?2, tumor necrosis factor?alpha ( TNF?α) , interleukin?1 beta ( IL?1β) and IL?6 mRNA expression by real?time PCR. Results There was no significant difference in thermal paw withdrawal latency at each time point between groups. Compared with C and S groups, MWT at T1-4 in NP and TRESK groups and at T1-3 in Virus and NS groups were significantly decreased, and the expression of MCP?1, MIP?2, TNF?α, IL?1βand IL?6 mRNA was up?regulated in NP, TRESK, Virus and NS groups. Compared with group NP, MWT was significantly increased at T1-4, and the expres?sion of MCP?1, MIP?2, TNF?α, IL?1β and IL?6 mRNA was down?regulated in group TRESK. Conclusion The mechanism by which intrathecal TRESK gene recombinant adenovirus reduces NP is re?lated to inhibition of inflammatory responses mediated by chemokine in the spinal cord of rats.

Objective To evaluate the methodological performance of the new enzymatic method for detecting glycated hemo-globin(HbAIc)and its influencing factors.Methods HbAIc was detected by the enzymatic method.The precision,anti-interfer-ence,recovery rate,accuracy and the influence of pre-processing(anti-coagulation,preservation,centrifugation)on the detection re-sults were evaluated,its correlation with HPLC and the bias degree were analyzed.Results The within-run coefficients of variation (CVs)for high,middle and low value QC samples in the enzymatic assay were 1.04%,1.26% and 1.37% respectively and the be-tween-run CVs were 1.83%,2.24% and 2.64%,respectively;the enzymatic method showed the linear correlation with HPLC(r=0.996,P <0.01);the HbA1c target value concentrations were 5.20%,6.40%,7.60%,8.80%,10.00% and 11.20% respectively, the recovery rates were 100.15%,98.91%,98.84%,98.20%,103.62% and 99.82% respectively;the interference test showed that this method had no significant interference on the detection results when glucose <15.50 mmol/L,UA<516.00 μmol/L,bili-rubin <217.00 μmol/L,triglyceride<10.20 mmol/L,urea<11.50 mmol/L,albumin<50 g/L and globulin <50 g/L.The HbA1c detection results in the samples with anti-coagulation by heparin sodium,EDTA-2K and sodium citrate stored for 3 d under -20-20 ℃ had no obvious change (P >0.05);the sample was centrifuged at 500,1 000 r/min(R=15 cm)for different time(1,2,5,10 min)and at 2 000 r/min for 1 min,their detection results had statistical differences compared with the sample centrifuged=3 000 r/min for 5 min (P <0.05).Conclusion The precision,anti-interference,accuracy and linearity range of the enzymatic method all conform to the clinical requirement.Compared with the conventional method,its correlation is good with small deviation,which can entirely satisfy the demand of the HbAIc detection in clinic.