Objective To observe the effects of ultrashort wave diathermy on the expression of tumor necrosis factor-α (TNF-α) andBcl-2 in hippocampus, striatum and motor cortex of rats with cerebral ischemia/reperfusion. Methods The model of focal ischemia/reperfusionin Sprague-Dawley rats was induced with intraluminal middle cerebral artery occlusion (MCAO) with nylon monofilament suture. Therats were divided into 3 groups: sham group (n=6), model group (n=6) and ultrashort wave diathermy group (n=6). Brain tissue slices wereimmunohistochemically stained (SABC) to observe the expression of TNF-α and Bcl-2. Results The expression of TNF-α and Bcl-2 in lefthippocampus, striatum and motor cortex was higher in the model group than in the sham group (P<0.01). The expression of TNF-α was lowerbut Bcl-2 was higher in the ultrashort wave diathermy group than in the model group (P<0.05). Conclusion Ultrashort wave diathermycan affect the expression of TNF-α and Bcl-2, which may associate to the neuroprotection from focal cerebral ischemia/reperfusion injury.

Background Retinal hypoxia is one of primary causes of retinal neovascularization,and its mechanism is research hot topic.Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial cells,which leads to angiogenesis.Besides to the vascular endothelial growth factor,the effect of adenosine on angiogenesis is increasingly concerned.Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial cells (HMEC-1) under the hypoxia and adenosine,and to explore the effect of adenosine on angiogenesis under hypoxia.Methods HMEC-1 cell line was cultured in vitro,and the cells were divided into normoxia group and hypoxia group.The cells in the normoxia group were cultured under the 5％ CO2 environment,and those in the hypoxia group were cultured under the 1％ O2,94％ N2 and 5％ CO2 environment.The proliferation ability and percentage of the cells were assayed by cell counting kit-8 (CCK8) and EDU.The migration number and invasive number of the cells were detected by transwell chamber.The expressions of CD39 and CD73 proteins in the cells were tested by Western blot and immunofluorescence technique,and the level of adenosine was measured by high performance liquid chromatography (HPLC).Results The proliferation values (absobancy) were 0.715-±0.067 and 0.821 ±0.056 in the normoxia group and the hypoxia group in 12 hours after culture,and those in 24 hours were 0.946±0.028 and 0.998±0.028,showing significant increase in the hypoxia group compared with the normoxia group (t12h =3.805,t24h =3.222,all at P ＜ 0.01).The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group (t =-6.868,P＜0.01).The number of the cell migration and invasion in 24 hours after culture was 185.3 ± 10.594 and 74.2± 10.741 respectively in the normoxia group,and that in the hypoxia group was 300.7±22.853 and 107.5±7.007,with significant differences between the two groups (t=-12.124,-6.367,both at P＜0.01).The expression levels of CD39 and CD73 proteins in the hypoxia group were significantly higher than those in the normoxia group in bothl2 hours and 24 hours after culture(all at P＜0.05),and the adenosine content in the cells is gradually increased in 2,6,12,24 and 36 hours after culture,with the highest content in 36 hours.The adenosine content was significantly higher in the hypoxia group than in the normoxia group at various time points (t2h =2.469,P =0.017;t6h =5.442,P＜0.001;t12h =3.841,P＜0.001;t24h =4.458,P＜0.001;t36h =2.757,P =0.008;t48h =3.319,P =0.002).Conclusions Compared with the normoxia group,the proliferation,migration and invasion abilities of HMEC-1 are stronger,meanwhile,the expression of CD39 and CD73 as well as the adenosine level in the cells are all increased under the hypoxic condition.It is suggested that the activation of human microvascular endothelial cells might be significantly related to the level of adenosine and its key enzymes.

Objective To explore the effect of tumor necrosis factor-alpha(TNF-α) blockade therapy on circulating Th17 cell percentage and serum interleukin (IL)-17 level in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Methods Twenty-seven RA and 22 AS patients were recruited, of which 14 cases from both diseases received 40 weeks TNF blockade therapy. Twenty-four healthy blood donors were used as controls. The frequencies of circulating Th17 cells were determined by flowcytometry, and serum IL-17 level were measured by enzyme linked immunosorbent assay(ELISA). Results Significantly higher baseline circulating Th17 cells were observed in active RA and AS patients compared with the healthy controls[RA 1.03%(0.66%,1.78%) vs controls 0.50%(0.43%,0.67%), Z=-3.236, P<0.01; AS(1.16±0.09)%vs controls (0.59 ±0.061)% , t =5.226, P <0.01]. Similarly, serum IL-17 level were significantly elevated in patients with both diseases compared with controls[RA(32.3±2.5) pg/ml vs controls(14.3±2.5) pg/ml, t=5.070, P<0.01; AS 28.98(23.84,36.14) pg/ml vs controls 11.84(5.33,22.12) pg/ml, Z=-4.103, P<0.01]. After TNF-α blockade therapy, serum IL-17 was significantly decreased in both diseases groups[RA △(-13.5± 5.0) pg/ml and AS △(-16.0±1.9) pg/ml]. In contrast, no significant differences were found in the frequencies of circulating Th17 cells[RA △(0.104 5±0.212 6)% and AS △(0.002 5±0.183 8)%]. Conclusion Th17 cells and IL-17 have been implicated in the pathogenesis of RA and AS. TNF-α blockade can partially inhibit the function of Th17 cells. However, it is unable to reduce the frequencies of these cells in the circulation after 40 weeks therapy, which may explain the reasons for the relapse.

Objective To investigate the value of fluid resuscitation strategy in septic shock patients by pulse indicator continuous cardiac out ( PiCCO ) .Methods 42 septic shock patients were divided into the PiCCO group(n=26) and the control group(n=16).All patients measured heart rate(HR),mean artery pressure(MAP), central venous pressure(CVP);CI,GEDVI,SVRI,EVLWI,CVP as indicator of fluid resuscitation after 0h,6h,24h of the diagnosis were measured respectively in PiCCO group;CVP as guiding volume resuscitation was measured in the control group .The effect of fluid resuscitation was compared between two groups .To analyse the relationship between CVP,GEDVI and CI in PiCCO group .according to CVP increase 2mmHg ,GEDVI whether elevated 10%.Results After 6h EGDT treatment bundle HR ,MAP,APACHEII score and clearance rate of lactic in PiCCO group improved more than those in control group [(101.3 ±7.8) and (119.4 ±7.2),t=-7.520,P<0.05;(71.8 ±7.6) and (51.5 ±8.9),t=7.873,P<0.05;(17.0 ±3.4) and (22.7 ±4.1),t=-4.978,P>0.05;(53.6 ±11.5) and (-16.5 ±5.2),t =9.283,P <0.05].There were no differences in 28-Day mortality between two groups (t =-2.162,P>0.05),but ICU hospitalization time decreased in PiCCO group [(13.8 ±2.6) and (23.3 ±2.2),t=-5.075,P<0.05].Changes in GEDVI was positively correlated with Changes in CI (r=0.799,P<0.05),while changes in CVP was poorly correlated with CI (r=-0.446,P>0.05).Conclusion Hemodynamic monitoring by PiCCO directed fluid resuscitation strategy can elevate reversal rate .Compared with pressure index CVP ,GEDVI is a sensitive indicator of cardiac preload .Correlation between CVP and GEDVI can reflect cardiac function ,Especially for septic shock patients with cardiac depression .

Cytokinesis ; Micronucleus Tests

Objective To study the structure of class 1 integrons in 90 strains of Pseudomonas aeruginosa isolated during two periods of 1992-1996 and 2003-2005,and to get information about the structure changing of class 1 integrons by comparing their structures in two different periods.Methods Routine PCR and long PCR were performed to amplify the class 1 integrons and the gene cassettes they carried, followed with sequencing and blast via GenBank.Results Thirteen out of 41 strians ioslated during the period of 1992-1996 were positive on class 1 intergrons.Long PCR showed that the class 1 integron was 1868 bp in length and contained 2 resistance genes averagely.Six types of resistance genes of qacEA1 (n=6), sull (n=14),aadA1 (n=2),aadB (n=1),PSE-1 (n=2) and tetA (n=1) were found in these integrons, which consisted of 5 patterns of resistance cassette arrangements.Nineteen strains were proved to carry class 1 integrons in 49 isolates from 2003-2005.The mean DNA sequence length of them was 3383 bp with 3.6 resistant genes in averagely,10 types of resistance genes,qacEA1 (n=18),sull (n=25),aadA1 (n=6), aadB (n=7),aacA4 (n=2),PSE-1 (n=3),VEB-1 (n=4),OXA10 (n=1),cm1 A (n=1) and tetA (n =2),were identified in these integrons,which were composed of 9 patterns of resistance cassette arrangements.Conclusion In terms of produce length and resistance cassettes carried in the integrons, greater complexity is found in the structure of class 1 integrons in strains isolated during 2003-2005 than those during 1992-1996.

Objective To study the relationship among apoptosis,NF-KB activation and uPA expres- sion in human colon carcinoma cell line HCTll6 induced by 5-fluorouracil,and to observe the effect of in- hibiting activity of NF-KB by PDTC on apoptosis as well as expression of uPA.Methods Cell apoptosis was analysed by Annexin V-FITC.Fluctuation of NF-KB and uPA was detected by semi-quantitative immuno- histochemistry.Results 5-fluorouracil could induce apoptosis and activate NF-KB.PDTC could significantly increase the apoptosis and suppress the activation of NF-KB induced by 5-fluorouracil.There was a positive correlation between the changes of uPA and NF-KB.Conclusion 5-fluorouracil could induce apoptosis,ac- tivate NF-KB and up-regulate expression of uPA of HCT116 cells.The mechanism of enhanced apoptosis by PDTC may be related to suppressing activation of NF-?B and down-regulating expression of uPA.

Objective To compare the sub-cellular proteome of isoniazid ( INH)-resistant Mycobacterium tuberculosis (MTB) with that of sensitive strains for identifying of unique proteins of these strains and discussing their preliminary application in clinical diagnosis. MethodsProteins of cell wall and membrane of 5 INH-resistant strains and 5 INH-sensitive strains were extracted by density gradient centrifugation.The extracts were subsequently analyzed using weak cation exchange (WCX) liquid chromatography ( LC )followed reverse phase (RP) liquid chromatography to compare the sub-cellular protein patterns. A total of 1280 fractions were collected and identified by matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF MS/MS). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for cell component and biological process analysis. Normalized Spectral Abundance Factors (NASF) was used for semi-quantity of protein expression. 5 proteins significantly up-regulated in INH-resistant strains and 2 proteins significantly up-regulated in INH-sensitive strains were selected for ELISA analysis with autologous sera respectively. Results A total of 347 proteins were identified. Cell component analysis showed that 58％ proteins were cells well or membrane proteins. Biological process analysis showed that 31％ proteins involved in carboxylic/monocarboxylic acid biosynthetic and metabolic process, 26％ and 15％ proteins involved in organic acid or fatty acid biosynthetic and metabolic process,while 28％ proteins involved in lipid biosynthetic , metabolic, transport and localization process. O-succinylbenzoate synthase, monooxygenase, hypothetical protein Rv2255c, nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase and membrane phosphatidate cytidylyltransferase cdsA were up-regulated in INH-resistant strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-sensitive sera. The differences between the INH-resistant sera and the INH-sensitive sera were significant ( t = 0.028, 0.044, 0.066, 0.064, 0.083, all P＜0.01 ). Chain A of Rv2002 Gene Product and Chain A of Crystal Structure Of Rv2632c were up-regulated in INH-sensitive strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-resistant sera. The differences between the INH-sensitive sera and the INH-resistant sera were significant (t=0.053, 0.073, both P＜0.05). ConclusionThe combination of density gradient centrifugation and 2D-LC MS/MS technology is useful in enrichment and identification of differential expressed proteins between INH-resistant and INH-sensitive strains at sub-cellular level. It is useful in finding antigens associated with INH-resistant MTB infection, which may prove useful for further study in the mechanism of INH resistant, as well as interaction between MTB and host.

AIM: To identify the non-steroid transcription factors upregulating the expression of L-plastin in hormone-independent prostate cancer, and partly elucidate the mechanism of hormone-refractory prostate cancer. METHODS: TF SEARCH software was used to analysis the possible binding sites of transcription factors in the 3’ end of L-plastin promoter that had been identified as important part of regulation response elements. Gel shift assay and supershift assay were used to confirm the transcription factors binding the speculated response elements. PCR site-mutagenesis technique was performed to delete the binding site of transcription factor and luciferase activity assay was carried out after deletion of the binding site. RESULTS: SP-1 respond element GGTGGGGCGGGGA located at -54- -41 of L-plastin promoter was identified with the TF SEARCH software. Gel shift assay and supershift assay confirmed that SP-1 was the transcription factor binding to GGTGGGGCGGGGA. Mutant deleted the SP-1 binding-site had low-luciferase activity than that of the naive. CONCLUSION: SP-1 plays an important role in the up-regulation of L-plastin expression in hormone-independent prostate cancer.

Objective To investigate the prediction function of preoperative B-type natriuretic peptide in patients to receive on-pump coronary artery bypass grafting with postoperative complications. Methods One hundred and thirty-two patients , including 78 males and 54 females , received on-pump coronary artery bypass grafting from January 2013 to November 2014 and were enrolled in the study. The patients were (63 ± 11.35) years old ( range from 35 to 82 years). The level of BNP was determined before operation, after operation, and on day 1, 2, 3 and 7 post-operation. Relationships were analyzed between BNP and LVEF,ventilation time, length of stay in ICU, the need for inotropic agents or intra-aortic balloon pump (IABP), incidence of postoperative atrial fibrillation, and acute renal failure. Receiver operating characteristic (ROC) curve analysis was also performed to predict the role of BNP in postoperative complications. Result A negative correlation between preoperative BNP level and preoperative LVEF(r = -0.512,P < 0.05) was found. The preoperative BNP level was positively correlated with a series of adverse events. The preoperative BNP was used to predict incidence of postoperative atrial fibrillation , the possibility of using IABP , renal failure , length of stay in ICU exceeding 48h or mortality at 28 days, and the area under the ROC curve (AUC) was 0.780, 0.893, 0.818 and 0.820, respectively. Conclusion The preoperative BNP level is well correlated with the cardiac function before CABG , which may be a good predictor of postoperative complications after CABG.